Intelligent Design, the best explanation of Origins

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The DNA double helix - evidence of design

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1 The DNA double helix - evidence of design on Sun May 24, 2015 12:12 pm


The DNA double helix, evidence of design

Formation of Deoxyribonucleotides
RNR Mechanism  and reaction
Is the diiron(III) cluster of ribonucleotide reductase reduced and oxidized during turnover?
RNR Class III [4Fe-4S] cluster and AdoMet biosynthesis
How Are Thymine Nucleotides Synthesized?
Prebiotic cytosine synthesis
Prebiotic thymine synthesis

Nucleotides are the constituents of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), the molecular repositories of genetic information. The ability to store and transmit genetic information from one generation to the next is a fundamental condition for life. The amino acid sequence of every protein in a cell, and the nucleotide sequence of every RNA, is specified by a nucleotide sequence in the cell’s DNA. A segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA, is referred to as a gene. The storage and transmission of biological information are the only known functions of DNA.

Fine-tuned regulation of nucleotide metabolism to ensure DNA replication with high fidelity is essential for proper development in all free-living organisms 15

What Are the Structure and Chemistry of Nitrogenous Bases?
The bases of nucleotides and nucleic acids are derivatives of either pyrimidine or purine. Pyrimidines are six-membered heterocyclic aromatic rings containing two nitrogen atoms (Figure 10.2a). The atoms are numbered in a clockwise fashion, as shown in Figure below: 

(a) The pyrimidine ring system; by convention, atoms are numbered as indicated. 
(b) The purine ring system, atoms numbered as shown.

The purine ring system consists of two rings of atoms: one resembling the pyrimidine ring and another resembling the imidazole ring a (Figure b). The nine atoms in this fused ring system are numbered according to the convention shown. The pyrimidine ring system is planar, whereas the purine system deviates somewhat from planarity in having a slight pucker between its imidazole and pyrimidine portions. Both are relatively insoluble in water, as might be expected from their pronounced aromatic character. 9

Three Pyrimidines and Two Purines Are Commonly Found in Cells
The common naturally occurring pyrimidines are cytosine, uracil, and thymine (5-methyluracil) (Figure below).

The common pyrimidine bases—cytosine, uracil, and thymine—in the tautomeric forms predominant at pH 7.

Cytosine and thymine are the pyrimidines typically found in DNA, whereas cytosine and uracil are common in RNA. Note that the 5-methyl group of thymine is the only thing that distinguishes it from uracil. Various pyrimidine derivatives, such as dihydrouracil, are present as minor constituents in RNA molecules. Adenine (6-amino purine) and guanine (2-amino-6-oxy purine), the two common purines, are found in both DNA and RNA (Figure below).

The common purine bases—adenine and guanine—in the tautomeric forms predominant at pH 7.

The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature
The aromaticity of the pyrimidine and purine ring systems and the electron-rich nature of their carbonyl and ring nitrogen substituents endow them with the capacity to undergo keto–enol tautomeric shifts b. That is, pyrimidines and purines exist as tautomeric pairs c . The keto tautomers of uracil, thymine, and guanine vastly predominate at neutral pH. In other words, pKa values d for ring nitrogen atoms 1 and 3 in uracil are greater than 8 (the pKa value for N-3 is 9.5). In contrast, the enamine form of cytosine predominates at pH 7 and the pKa value for N-3 in this pyrimidine is 4.5. Similarly, for guanine, the pKa value is 9.4 for N-1 and less than 5 for N-3. These pKa values specify whether protons are associated with the various ring nitrogens at neutral pH. As such, they are important in determining whether these nitrogens serve as H-bond donors or acceptors. Hydrogen bonding between purine and pyrimidine bases is fundamental to the biological functions of nucleic acids, as in the formation of the double-helix structure of DNA. The important functional groups participating in H-bond formation are the amino groups of cytosine, adenine, and guanine; the ring nitrogens at position 3 of pyrimidines and position 1 of purines; and the strongly electronegative oxygen atoms attached at position 4 of uracil and thymine, position 2 of cytosine, and position 6 of guanine. 

Nucleotides are building blocks for DNA and RNA. These molecules consist of three components: a phosphate, a ribose sugar, and a nitrogenous (nitrogen-containing) ring compound that behaves as a base in solution (a base is a substance that can accept a proton in solution). Nucleotide bases appear in two forms: A single-ring nitrogenous base, called a pyrimidine, and a double-ringed base, called a purine. There are two kinds of purines (adenine and guanine) and three pyrimidines (uracil, cytosine, and thymine). Uracil is specific to RNA, substituting for thymine. In addition, RNA nucleotides contain ribose, whereas DNA nucleotides contain deoxyribose (hence the origin of their names). Ribose has a hydroxyl (OH) group attached to both the 2′ and 3′ carbons, whereas deoxyribose is missing the 2′ hydroxyl group. 6

Nucleotide metabolism is central to all biological systems, due to their essential role in genetic information and energy transfer, which in turn suggests its possible presence in the last common ancestor (LCA) of Bacteria, Archaea and Eukarya. [url=Nucleotide metabolism is central to all biological systems, due to their essential role in genetic information and energy transfer, which in turn suggests its possible presence in the last common ancestor (LCA) of Bacteria, Archaea and Eukarya.]5[/url]

Example of a ribonucleotide- guanonsine monophosphate

DNA is the “blueprint of life” and stores within the necessary instructions for living cells to grow and to function. The existence of DNA has been known since 1869. It took, however, almost a century to discern DNA structure and its role in the storage of genetic information. Cellular DNA undergoes harmful modifications every day as a result of exposure to UV light, environmental stress, and toxic chemicals. DNA damage can also result from errors during DNA synthesis. Damaged DNA must be repaired promptly and efficiently; otherwise, the replication machinery can incorporate the wrong nitrogenous base, leave nicks and gaps, and stall or disengage during subsequent rounds of DNA synthesis, resulting in deleterious mutations and chromosomal instability. The cell utilizes a number of repair pathways to prevent the loss of genetic information. The enzymes that are involved in the repair process are specific to the type of DNA damage encountered and depend on the stage of the cell cycle. Not surprisingly, defects in key components of these systems in humans are associated with a broad spectrum of disorders, usually characterized by premature aging, susceptibility to cancers, and other diseases bearing hallmarks of aging, immunodeficiency, or mental retardation.

It's evident that DNA repair mechanisms are essential for cells to function and to survive. The DNA repair mechanisms could not have evolved after life arose but must have come into existence before. The mechanisms are highly complex and elaborated, as consequence, the design inference is justified and seems to be the best way to explain its existence.

DNA is something like a computer tape that stores many programs for a large computer to run. If we would scale up the linear dimension of DNA by a factor of 1 000 000 or 10^6. When we do so, the relative sizes and proportions of objects remain the same. Note that the length of DNA from a typical chromosome on this expanded scale is about 30 km, while its diameter is just 2 mm. Very few objects in the physical world are so long and so narrow.

Following the unresolved issues of nucleotide biogenesis :

(1) Laboratory experiments show that DNA spontaneously and progressively disintegrates over time. Estimates indicate that DNA should completely break down within 10,000 years. Any fossil DNA remaining after this period (especially more than say 100,000 years) must of necessity indicate that the method of dating the fossil is in error. Nature, Vol. 352, August 1, 1991 p:381

(2) The classic evolutionary problem of 'which came first, protein or DNA' has not been solved by the 'self-reproducing' RNA theory as many textbooks imply. The theory is not credible as it was based on laboratory simulations which were highly artificial, and were carried out with a 'great deal of help from the scientists'. Scientific American, February, 1991 p:100-109

(3) DNA can only be replicated in the presence of  specific enzymes which can only be manufactured by the already existing DNA. Each is absolutely essential for the other, and both must be present for the DNA to multiply. Therefore, DNA has to have been in existence in the beginning for life to be controlled by DNA. Scott M. Huse, "The Collapse of Evolution", Baker Book House: Grand Rapids (Michigan), 1983 p:93-94

(4) There is no natural chemical tendency for the series of base chemicals in the DNA molecule to line up a series of R-groups in the orderly way required for life to begin. Therefore being contrary to natural chemical laws, the base-R group relationship and the structure of DNA could not have formed by random chemical action. Scott M. Huse, "The Collapse of Evolution", Baker Book House: Grand Rapids (Michigan), 1983 p:94

(5) "The origin of the genetic code is the most baffling aspect of the problem of the origins of life and a major conceptual or experimental breakthrough may be needed before we can make any substantial progress." Written by biochemist Dr Leslie Orgel (Salk Institute, California) in the article "Darwinism at the Very Beginning of Life" in New Scientist, April 15, 1982 p:151

(6) Computer scientists have demonstrated that information does not, and cannot arise spontaneously. Information only results from the input of energy, under the all-important direction of intelligence. Therefore, as DNA is information, it cannot have been formed by natural chemical means. P. Moorhead & M. Kaplan (eds.), "Mathematical Challenges to the Neo-Darwinian Interpretation of Evolution", Wistar Institute: Philadelphia (Pennsylvania), 1967

(7) The transformation of one species into another by viruses transferring small sections of the DNA of another species could not cause evolution for three reasons:- (1) if genes for a particular feature or action were transmitted as a small piece of DNA, the animal would not be able to utilize the code unless it had all the other structures present to support that feature, (2) there is no guarantee that without the rest of the supporting DNA code, that the feature would appear in the right place, and (3) the information transmitted would already be in existence and would not lead to the formation of a species with totally new features. Reader's Digest, March 1980

(8  "A scientist who won the Nobel Prize for his discovery of the DNA technique that inspired (the film) Jurassic Park was asked how likely it was that in the future, a dinosaur could be re-created from ancient DNA trapped in amber, as in the movie. Dr Kary Mullis replied in essence that it would be more realistic to start working on a time machine to go back and catch one." From Creation Ex Nihilo, Vol. 16, No. 2, March 1994, p:8, summarizing The Salt Lake Tribune, December 5, 1993

Molecular Biology: Principles of Genome Function page 61

Identifying conditions that lead to the robust synthesis of nucleic acids has been much more difficult. First, there are several chemically distinct components that are needed:
the nucleotide bases, the sugar moieties, and the phosphate backbone. Although adenine is synthesized efficiently from mixtures of hydrogen cyanide and ammonia, the other bases (G, C, and U) are much less readily synthesized. Prebiotic synthesis of the sugar ring, ribose, presents another significant chemical challenge, as does formation of the glycosidic bond between the bases and the sugars.

the basics :

evidence from biochemistry does not provide many clues to explain the evolution of pyrimidine and purine synthesis. 4

Evolution 1

It is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes. This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution. Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old,but these claims are controversial. Building blocks of DNA (adenine, guanine and related organic molecules) may have been formed extraterrestrially in outer space. Complex DNA and RNA organic compounds of life, including uracil, cytosine and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar dust and gas clouds.

The individual macromolecules are complex
But the complex interaction of biological macromolecules is only one aspect of the problem facing the origin of life. What compounds the enigma is that the individual macromolecular components are themselves complex, in the sense that their sequences - of ribonucleotides in the case of RNA, or amino acids for proteins - are very specific.
The linear amino acid sequence of a protein is specific because it must (a) be able to fold into a discrete 3-dimensional structure, and (b) have the right amino acids in the right positions in the linear sequence so that, when folded, they are in exactly the right positions in relation to each other to form the active site(s) of the protein. (And similar considerations apply to RNAs.)
Sequences which meet these criteria are exceedingly rare compared with the astronomical number of possible sequences of a suitable length. For example Douglas Axe has estimated that only 1 in about 10^74 possible sequences will have biological function (Axe). So it is totally unrealistic to think that such sequences could have arisen by chance. How much less a suite of mutually dependent macromolecules?
If the components themselves were not so improbable then it might be realistic to think that a complex combination of components could arise by chance; but the extreme improbability of the individual components is such that they are very unlikely to arise individually, and hence there is no chance whatever of an interdependent system.

Where even just two macromolecules are required to perform a function, then it would be necessary for both components to arise together: Because natural selection does not have foresight: if one component arises alone it will not be retained for potential future usefulness (when the second component is available), but will almost certainly degrade by mutation. And, it should be noted, if the probability of getting one component is 1 in 10^74 then the probability of getting two together is 1 in 10^148 (not 1 in 2x10^74); and so on for multi-component systems. This is why the obligatory mutual dependence of many macromolecules in even basic biological systems completely defies any hope of an evolutionary origin.
So, in summary, the crux of the problem is that even a basic biological replicating system requires (a) several macromolecules with complementary functions with (b) each having a highly improbable sequence. And this combination of complexities presents an insurmountable challenge to a naturalistic origin of life. 3

However, just as there are severe problems with an abiotic origin of polypeptides, similar issues apply to the production of polynucleotides, except that chemical considerations make the situation even worse.

This is because the nucleotides themselves each comprise a base, sugar, and phosphate (Figure 4) which need to be joined together correctly - involving two endothermic condensation reactions (with all the problems that means) to make a nucleotide in addition to the endothermic condensation reaction involved in joining the nucleotides. In other words, compared with polypeptides, nucleotides are even harder to synthesise and easier to destroy; in fact, to date, there are no reports of nucleotides arising from inorganic compounds in primeval soup experiments.

The origin of following must be explained :

the origin and synthesis of nucleotides :  

adenine (A) - a purine
cytosine(C) - a pyrimidine
guanine (G) - a purine
thymine (T) - a pyrimidine

- the formation of the double-helix spiral staircase-like structure
- why they are  running in opposite directions
- the backbone made up of (deoxyribose) sugar molecules
- the phosphate groups which links it together. ( also called 3'-5' phosphodiester linkage )
- the assembly and synthesis of the first structure

Scientists have long known that a myriad of sugars and numerous other nucleobases could have conceivably become part of the cell’s information storage medium (DNA). But why do the nucleotide subunits of DNA and RNA consist of those particular components? Phosphates can form bonds with two sugars simultaneously (called phosphodiester bonds) to bridge two nucleotides while retaining a negative charge. This makes this chemical group perfectly suited to form a stable backbone for the DNA molecule. 2

How is that better explained? Through natural processes, or intentional design?

Other compounds can form bonds between two sugars but are not able to retain a negative charge. The negative charge on the phosphate group imparts the DNA backbone with stability, thus giving it protection from cleavage by reactive water molecules. Furthermore, the intrinsic nature of the phosphodiester bonds is also finely-tuned. For instance, the phosphodiester linkage that bridges the ribose sugar of RNA could involve the 5’ OH of one ribose molecule with either the 2’ OH or 3’ OH of the adjacent ribose molecule. RNA exclusively makes use of 5’ to 3’ bonding. As it turns out, the 5’ to 3’ linkages impart far greater stability to the RNA molecule than does the 5’ to 2’ bonds.
Why do deoxyribose and ribose serve as the backbone constituents of DNA and RNA respectively? Both are five-carbon sugars which form five-membered rings. It is possible to make DNA analogues using a wide range of different sugars that contain four, five and six carbons that can form five- and six-membered rings. But these DNA variants possess undesirable properties as compared to DNA and RNA. For instance, some DNA analogues do not form double helices. Others do, but the nucleotide strands either interact too tightly or too weakly, or they display inappropriate selectivity in their associations. Furthermore, DNA analogues made from sugars that form 6-membered rings adopt too many structural conformations. In this event, it becomes exceptionally difficult for the cell’s machinery to properly execute DNA replication and transcription. Other research shows that deoxyribose uniquely provides the necessary space within the backbone region of the double helix of DNA to accommodate the large nucleobases. No other sugar fulfils this requirement.

The right properties of deoxyribose and ribose are in my view far better explained through a designer, than random natural processes.

The molecular constituents of the DNA structure appear to have optimized chemical properties to produce a stable helical structure capable of storing the information required for the cell’s operation. Detailed accounts of how such an optimized structure for the cell’s most fundamental information storage medium could have arisen naturally have not been produced. To suppose that such extensive optimization could have come into being by blind chance is a far greater leap of faith than design.

If there is no salt in the surrounding medium, there is a strong repulsion between the two strands and they will fall apart. Therefore counter-ions are essential for the double-helical structure.

- the origin of the counter-ions

Nucleotide biosynthesis

Purines and pyrimidines are derived largely from amino acids.  The amino acids glycine  and aspartate  are the scaffolds on which the ring systems present in nucleotides are assembled. Furthermore, aspartate and the side chain of glutamine serve as sources of NH2 groups in the formation of nucleotides. In de novo (from scratch) pathways, the nucleotide bases are assembled from simpler compounds. The framework for a pyrimidine base is assembled first and then attached to ribose. In contrast, the framework for a purine base is synthesized piece by piece directly onto a ribose-based structure. These pathways each comprise a small number of elementary reactions that are repeated with variation s to generate different nucleotides. 13

De novo pathways lead to the synthesis of ribonucleotides. However, DNA is built from deoxyribonucleotides. Consistent with the notion that RNA preceded DNA, all deoxyribonucleotides are synthesized from the corresponding ribonucleotides. The deoxyribose sugar is generated by the reduction of ribose within a fully formed nucleotide. Furthermore, the methyl group f that distinguishes the thymine of DNA from the uracil of RNA is added at the last step in the pathway. A nucleoside is a purine or pyrimidine base linked to a sugar and that a nucleotide is a phosphate ester of a nucleoside e

A nucleoside is a purine or pyrimidine base linked to a sugar and that a nucleotide is a phosphate ester of a nucleoside. 

dATP (desoxiAdenosine Trifosfate)
dCTP (desoxiCitidine Trifosfate)
dGTP (desoxiGuanose Trifosfate)
dTTP (desoxiTimidine Trifosfate)

Biosynthesis of the Ribose 5-phosphate Ring
The starting material for purine biosynthesis is Ribose 5-phosphate, a product of the pentose phosphate pathway. In contrast to purine synthesis, the pyrimidine ring system is constructed before a ribose-5-phosphate moiety is attached.  Ribose 5-phosphate (R5P) is both a product and an intermediate of the pentose phosphate pathway. The last step of the oxidative reactions in the pentose phosphate pathway is the production of ribulose 5-phosphate.  R5P is produced in the pentose phosphate pathway in all organisms. When more R5P is needed than NADPH, R5P can be formed through glycolytic intermediates.  During nucleotide biosynthesis, R5P undergoes activation by ribose-phosphate diphosphokinase (PRPS1) to form phosphoribosyl pyrophosphate (PRPP). Formation of PRPP is essential for both the de novo synthesis of purines and for the purine salvage pathway.  14


Imidazole is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole, and having non-adjacent nitrogen atoms. 8

b In organic chemistry, keto–enol tautomerism refers to a chemical equilibrium between a keto form (a ketone or an aldehyde) and an enol (an alcohol). The enol and keto forms are said to be tautomers of each other. The interconversion of the two forms involves the movement of an alpha hydrogen and the shifting of bonding electrons; hence, the isomerism qualifies as tautomerism. 10

c Tautomers are constitutional isomers of organic compounds that readily interconvert.[2][3][4] This reaction commonly results in the relocation of a proton. Tautomerism is relevant to the behavior of amino acids and nucleic acids, two of the fundamental building blocks of life. 11

d An acid dissociation constantKa, (also known as acidity constant, or acid-ionization constant) is a quantitative measure of the strength of an acid in solution. It is the equilibrium constant for a chemical reaction known as dissociation in the context of acid–base reactions 12

e Who had the "good idea" to exchange  Thymine to Uracil?

f A methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms — CH3. 14

6. The Cell, Panno, page 9
7. Origins of life : biblical and evolutionary models,  page 68
9. Biochemistry 6th. edition, Garrett, page 326
13. Biochemistry, 5th edition, Lubert Stryer page 734

Further readings:
Origin of life: instability of building blocks

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2 Pyrimidines on Sun May 24, 2015 5:30 pm


Pyrimidines 6

In nucleic acids, three types of nucleobases are pyrimidine derivatives: cytosine (C), thymine (T), and uracil (U). 6 

The Watson–Crick base pairs A;T and G;C

Synthesis of Pyrimidines  6

De novo pyrimidine synthesis pathway can be explained by the following steps. 30

Synthesis of Carbamoyl Phosphate
Synthesis of Carbamoyl Aspartate
Ring Closure to form dihydro orotate
Oxidation of Dihydro Orotate
Addition of ribose Phosphate moiety
De Carboxylation to form UMP

A schematic representation of the pyrimidine biosynthesis pathway.
CTP, the end product of the pathway, inhibits ATCase, which catalyzes the pathway’s first step.

Pyrimidine metabolism


The biosynthesis of pyrimidines is simpler than that of purines. Isotopic labeling experiments have shown that atoms N1, C4, C5, and C6 of the pyrimidine ring are all derived from aspartic acid, C2 arises from HCO-3, and N3 is contributed by glutamine.

In contrast to purines, pyrimidines are not synthesized as nucleotide derivatives. Instead, the pyrimidine ring system is constructed before a ribose-5-P moiety is attached. Also, only two precursors, carbamoyl-P and aspartate, contribute atoms to the six-membered pyrimidine ring compared to seven precursors for the nine purine atoms. Mammals have two enzymes for carbamoyl phosphate synthesis. Carbamoyl phosphate for pyrimidine biosynthesis is formed by carbamoyl phosphate synthetase II (CPS-II), a cytosolic enzyme. 

The metabolic origin of the six atoms of the pyrimidine ring.

UMP is Synthesized in Six Steps
The common pyrimidine ribonucleotides are cytidine 5'-monophosphate (CMP; cytidylate) and uridine 5'-monophosphate (UMP; uridylate), which contain the pyrimidines cytosine and uracil. Uridine monophosphate (UMP) a, which is also the precursor of Cytidylate ( CMP ), is synthesized in a six-reaction pathway. In contrast to purine nucleotide synthesis, the pyrimidine ring is coupled to the ribose-5-phosphate moiety after the ring has been synthesized.

Enzymes used in the process:

1. Carbamoyl phosphate synthase II
2. Aspartate carbamoyltransferase
3. Dihydroorotase
4. Dihydro Orotate Dehydrogenase
5. Orotate Phosphoribosyl transferase
6. Orotidine 5'-phosphate decarboxylase
7. Nucleoside-phosphate kinase  & Nucleoside-diphosphate kinase


The de novo synthesis of UMP ( Pyrimidine pathway ) 

De novo synthesis of pyrimidine nucleotides:
biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5- phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. The first step in this pathway is the synthesis of carbamoyl phosphate from CO2 and NH+4  , catalyzed in eukaryotes by carbamoyl phosphate synthetase II.

1.  Synthesis of carbamoyl phosphate. 
The first reaction of pyrimidine biosynthesis is the synthesis Carbamoyl phosphate of  from HCO-3 b and the amide nitrogen of glutamine by the cytosolic carbamoyl phosphate synthetase II  enzyme. 

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine.  27

Channeling of intermediates in bacterial carbamoyl phosphate synthetase. 
The large and small subunits are shown in gray and blue, respectively; the channel between active sites (almost 100 Å long) is shown as a yellow mesh. A glutamine molecule (green) binds to the small subunit, donating its amido nitrogen as NH4+ in a glutamine amidotransferase–type reaction. The NH4+enters the channel, which takes it to a second active  site, where it combines with bicarbonate in a reaction requiring ATP  (bound ADP in blue). The carbamate then reenters the channel to reach the third active site, where it is phosphorylated to carbamoyl phosphate  (bound ADP in red). 28

The carbamoyl-phosphate required in pyrimidine biosynthesis is made in the cytosol by Carbamoyl phosphate synthase II . In bacteria, a single enzyme supplies carbamoyl phosphate for the synthesis of arginine and pyrimidines. The bacterial enzyme has three separate active sites, spaced along a channel nearly 100 Å long (Figure above). Bacterial carbamoyl phosphate synthetase provides a vivid illustration of the channeling of unstable reaction intermediates between active sites.

This reaction consumes two molecules of ATP: One provides a phosphate group and the other energizes the reaction. Carbamoyl phosphate is also synthesized in the urea cycle. In that reaction, catalyzed by the mitochondrial enzyme carbamoyl phosphate synthetase I, ammonia is the nitrogen source.

The carbamoyl phosphate synthetase II (CPS-II) reaction.

Synthesis of carbamoyl phosphate
Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. 22  Nucleotides are building blocks of RNA. Both nucleotides and RNA are among the first biomolecules to arise in life supposedly long before DNA and proteins came into play. (Joyce, 2002). Since that time, nucleotide and nucleic acid metabolism have constituted a central part of the metabolism of every living organism.  Because of the early appearance  and the essential importance for life, nucleotide de novo synthesis is highly similar in almost all living organisms (Lehninger et al., 1994; Kafer et al., 2004). 23

The rate of cell growth establishes the basal rate of de novo pyrimidine biosynthesis, but the rapid changes in flux through the pathway requires precise metabolic control exerted by allosteric effectors and the activity of signaling cascades. 21

The pyrimidine synthesis is a similar process than that of Purines (Purines Synthesis). In the de novo synthesis of Pyrimidines, the ring is synthesized first and then it is attached to a ribose-phosphate to for a pyrimidine nucleotide. Pyrimidine rings are assembled from bicarbonate, aspartate, and Ammonia.

The biosynthetic pathway to pyrimidine nucleotides is simpler than that for purine nucleotides, reflecting the simpler structure of the base. In contrast to the biosynthetic pathway for purine nucleotides, in the pyrimidine pathway the pyrimidine ring is constructed before ribose-5-phosphate is incorporated into the nucleotide. The first pyrimidine mononucleotide to be synthesized is orotidine-5-monophosphate (OMP); and from this compound, pathways lead to nucleotides of uracil, cytosine, and thymine. OMP thus occupies a central role in pyrimidine nucleotide biosynthesis, somewhat analogous to the position of IMP in purine nucleotide biosynthesis. Like IMP, OMP is found only in low concentrations in cells and is not a constituent of RNA. 16

Pyrimidine rings are assembled from bicarbonate, aspartate, and ammonia. Although an ammonia molecule already present in solution can be used, the ammonia is usually produced from the hydrolysis of the side chain of glutamine. 10

Glutamate: An Amino Acid of Particular Distinction 11
The first reaction of Pyrimidine synthesis is the synthesis of Carbamoyl phosphate by utilizing amide form of Glutamine (Glutamate) and HCO3– (Carbonic acid).

Glutamate metabolism plays a vital role in the biosynthesis of nucleic acids and proteins. 9  Glutamate plays a pivotal role in the design of life processes  
The appearance of this and other amino acids on Earth arose in the far geological past, as many studies suggest, via abiotic synthesis

Of course, if the metabolic processes were inexistent before life began, it could not be different......

It is now quite apparent that the formation of amino acids occurs readily under inferred primitive earth conditions. Amino acids can be formed when various types of energies are applied to gas mixtures or from chemically reactive precursors; glutamic acid and its homolog, aspartic acid, have been detected when various sources of energy including electrical discharge, UV rays, ionizing radiation and thermal energy are introduced into such systems. Furthermore, organic compounds, including amino acids, have been thought to have appeared >3.5 × 109 y ago as a consequence of atmospheric shock waves within the early, strongly reducing atmosphere. 

Even IF that were true, how did Glutamate gain its homochirality? 

Melendez-Hevia et al. (1996) attempted to demonstrate that the evolution of the Krebs cycle emerged as a consequence of the opportunistic reorganizing and assembling of preexisting organic chemical reactions

This is remarkable. All they demonstrate is just so guesswork and claim that prebiotic molecules were looking for an opportunity for reorganizing.  Basically attributing teleology or goal orientation to non-conscient molecules.... that's as far as naturalistic explanations go. 

Now let's put that in contrast to glutamate metabolism in eukaryotes :

Finally, the central position of L-glutamate in mammalian metabolism (Fig.19.16 ) and, in particular, its presence in metabolic cycles, including the citric acid cycle, is noteworthy and should be brought into the prebiotic and evolutionary schema.

There is a huge, open, unexplained gap between the helpless explanation attempts and guesswork of the origin of Glutamate in a prebiotic world, and the staggering complexity observed today by modern cells to synthesize it.  In face of this picture, James Tour's note about today's status quo of abiogenesis research makes perfect sense :

" It is clear, chemists and biologists are clueless. I wrote, “Those who think scientists understand the issues of prebiotic chemistry are wholly misinformed. Nobody understands them. Maybe one day we will. But that day is far from today. It would be far more helpful (and hopeful) to expose students to the massive gaps in our understanding. "  

Summary and Conclusion
Because of their breadth, it may be that we have not entirely succeeded in addressing the issues posed to us at the outset.

Nice admission of ignorance. 

ATP: The  Energy  Currency for the Cell

ATP contains the purine base adenine and the sugar ribose which together form the nucleoside adenosine. Adenine is one of the most important organic molecules for life as we know it today - and it  would never accumulate in any kind of "prebiotic soup.

Adenine synthesis in a prebiotic earth

Adenine would never accumulate in any kind of "prebiotic soup.

Biochemistry, 8th ed. Styer, page 745:
The first step in de novo pyrimidine biosynthesis is the synthesis of Carbamoyl phosphate c from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed
by carbamoyl phosphate synthetase II (CPS II).

2: Synthesis of carbamoyl aspartate. 

Condensation of carbamoyl phosphate with aspartate to form carbamoyl aspartate is catalyzed by aspartate transcarbamoylase (ATCase). This reaction proceeds without ATP hydrolysis because carbamoyl phosphate is already “activated.” 

Aspartate transcarbamoylase catalyzes the formation of N-carbamoyl aspartate from carbamoyl phosphate and aspartate. 
Both of its substrates bind cooperatively to the enzyme. Moreover, ATCase is allosterically inhibited by cytidine triphosphate (CTP), a pyrimidine nucleotide, and is allosterically activated by adenosine triphosphate (ATP), a purine nucleotide.

Carbamoyl phosphate is condensing with Aspartic acid it forms carbamoyl aspartate is catalyzed by Aspartate Transcarbamoylase (ATCase).
Aspartate Transcarbamoylase ( ATCase ) EC


Aspartate carbamoyltransferase is a multi-subunit protein complex composed of 12 subunits

5 From: David Goodsell, Our Molecular Nature, page 26

Aspartate Carbamoyltransferase
Dozens of enzymes are needed to make the DNA bases cytosine and thymine from their component atoms. The second step is performed by aspartate carbamoyltransferase.  In bacteria, this enzyme controls the entire pathway. (In human cells, the regulation is more complex, involving the interaction of several of the enzymes in the pathway.) The enzyme is composed of six large catalytic subunits and six smaller regulatory subunits . The active site of the enzyme is located where two individual catalytic subunits touch, so the position of the two subunits relative to one another is critical. Take just a moment to ponder the immensity of this enzyme. The entire complex is composed of over 40,000 atoms, each of which plays a vital role. The handful of atoms that actually perform the chemical reaction are the central players. But they are not the only important atoms within the enzyme--every atom plays a supporting pan. The atoms lining the surfaces between subunits are chosen to complement one another exactly, to orchestrate the shifting regulatory motions. The atoms covering the surface are carefully picked to interact optimally with water, ensuring that the enzyme doesn't form a pasty aggregate, but remains an individual, floating factory. And the thousands of interior atoms are chosen to fit like a jigsaw puzzle, interlocking into a sturdy framework. Aspartate carbamoyltransferase is fully as complex as any fine automobile in our familiar world.

Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
The allosteric behavior of  ATCase has been investigated ( In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.)  and it was demonstrated that both of its substrates bind cooperatively to the enzyme. Moreover, ATCase is allosterically inhibited by cytidine triphosphate (CTP), a pyrimidine nucleotide, and is allosterically activated by adenosine triphosphate (ATP), a purine nucleotide. CTP  is a product of the pyrimidine biosynthetic pathway inhibits an earlier step in its own biosynthesis. Thus, when CTP levels are high, CTP binds to ATCase, thereby reducing the rate of CTP synthesis. Conversely, when cellular [CTP] decreases, CTP dissociates from ATCase and CTP synthesis accelerates. The metabolic significance of the ATP activation of ATCase is that it tends to coordinate the rates of synthesis of purine and pyrimidine nucleotides, which are required in roughly equal amounts in nucleic acid biosynthesis. For instance, if the ATP concentration is much greater than that of CTP, ATCase is activated to synthesize pyrimidine nucleotides until the concentrations of ATP and CTP become balanced. Conversely, if the CTP concentration is greater than that of ATP, CTP inhibition of ATCase permits purine nucleotide biosynthesis to balance the ATP and CTP concentrations.

So, if one of the effector molecules ( the on/off switch ) of Aspartate carbamoyltransferase is Cytidine triphosphate CTP, the very own nucleotide which it helps to produce. what came first ? The enzyme, or its product, which regulates the enzyme? And since ATP is required in the regulation process, had its synthesis not have to be fully setup as well ? 

The X-ray structure of ATCase reveals that the catalytic subunits are arranged as two sets of trimers in complex with three sets of regulatory dimers. Each regulatory dimer joins two catalytic subunits in different
c3 trimers.

X-Ray structure of ATCase from E. coli. 
The T-state enzyme in complex with CTP is viewed 
(a) along the protein’s molecular threefold axis of symmetry and 
(b) along a molecular twofold axis of symmetry perpendicular to the view in Part a. The polypeptide chains are drawn in worm form embedded in their transparent molecular surface. The regulatory dimers (yellow) join the
upper catalytic trimer (red) to the lower catalytic trimer (blue). CTP is drawn in space-filling form colored according to atom type (C green, O red, N blue, and P orange). 
(c) The R-state enzyme in complex with PALA viewed as in Part b. PALA (which is bound to the c subunits but largely obscured here) is drawn in space-filling form. Note how the rotation of the regulatory dimers in the T S R transition causes the catalytic trimers to move apart along the threefold axis.

Allosteric enzymes, such as ATCase, play a pivotal role in metabolism because they have three functions – they catalyze a unique metabolic reaction, alter the rate of catalysis in response to cellular conditions and are responsible for the rate of the larger pathway. Regulation of ATCase involves the binding of signaling molecules to the regulatory sites, and this binding induces an alteration in the rate of catalytic activity. Our studies of ATCase have been directed towards using the three-dimensional structural information to understand, on the molecular level, how ATCase is able to catalyze the reaction between aspartate and carbamoyl phosphate, how the enzyme is able to transition between the T and R conformations, and how the binding of regulatory nucleotides a distance of 60Å from the active site induces alterations to modulate activity. 29

Aspartate transcarbamoylase catalyzes the condensation of aspartate and carbamoyl phosphate to form N -carbamoylaspartate and orthophosphate. This reaction is the committed step in the pathway, which consists of 11 reactions, that will ultimately yield the pyrimidine nucleotides uridine triphosphate (UTP) and cytidine triphosphate (CTP). How is this enzyme regulated to generate precisely the amount of pyrimidines needed by the cell? ATCase is inhibited by CTP, the final product of the ATCase-initiated pathway. The rate of the reaction catalyzed by ATCase is fast at low concentrations of CTP but slows as CTP concentration increases. Thus, the pathway continues to make new pyrimidines until sufficient quantities of CTP have accumulated. The inhibition of ATCase by CTP is an example of feedback inhibition, the inhibition of an enzyme by the end product of the pathway. Feedback inhibition by CTP ensures that N-carbamoylaspartate and subsequent intermediates in the pathway are not needlessly formed when pyrimidines are abundant. The inhibitory ability of CTP is remarkable because CTP is structurally quite different from the substrates of the reaction. Thus CTP must bind to a site distinct from the active site at which substrate binds. Such sites are called allosteric or regulatory sites. CTP is an example of an allosteric inhibitor. In ATCase (but not all allosterically regulated enzymes), the catalytic sites and the regulatory sites are on separate polypeptide chains

ATCase consists of separable catalytic and regulatory subunits
What is the evidence that ATCase has distinct regulatory and catalytic sites? ATCase can be literally separated into regulatory (r) and catalytic (c) subunits.  The larger subunit is the catalytic subunit. The catalytic and regulatory subunits combine rapidly when they are mixed. The resulting complex has the same structure, as the native enzyme: two catalytic trimers and three regulatory dimers. Most strikingly, the reconstituted enzyme has the same allosteric and kinetic properties as those of the native enzyme. Thus, ATCase is composed of discrete catalytic and regulatory subunits, and the interaction of the subunits in the native enzyme produces its regulatory and catalytic properties. Two catalytic trimers are stacked one on top of the other, linked by three dimers of the regulatory chains ( See figure below )

Structure of (B) ATCase. 
(A) The quaternary structure of aspartate transcarbamoylase as viewed from the top. The drawing in the center is a simplified representation of the relations between subunits. A single catalytic trimer [catalytic (c) chains, shown in yellow] is visible; in this view, the second trimer is hidden below the one visible. Notice that each r chain interacts with a c chain through the zinc domain. 
(B) A side view of the complex.

There are significant contacts between the catalytic and the regulatory subunits: each r chain within a regulatory dimer interacts with a c chain within a catalytic trimer. The c chain makes contact with a structural domain in the r chain that is stabilized by a zinc ion bound to four cysteine residues. The zinc ion is critical for the interaction of the r chain with the c chain. The mercurial compound p-hydroxymercuribenzoate is able to dissociate the catalytic and regulatory subunits because mercury binds strongly to the cysteine residues, displacing the zinc and preventing interaction with the c chain. To locate the active sites, the enzyme is crystallized in the presence of  N  (phosphonacetyl)- L -aspartate (PALA), a bisubstrate analog (an analog of the two substrates) that resembles an intermediate along the pathway of catalysis ( Figure below )

PALA, a bisubstrate analog. 
(Top) Nucleophilic attack by the amino group of aspartate on the carbonyl carbon atom of carbamoyl phosphate generates an intermediate on the pathway to the formation of N-carbamoylaspartate. (Bottom) N-(Phosphonacetyl)-L-aspartate (PALA) is an analog of the reaction intermediate and a potent competitive inhibitor of aspartate transcarbamoylase.

PALA is a potent competitive inhibitor of ATCase that binds to and blocks the active sites. The structure of the ATCase– PALA complex reveals that PALA binds at sites lying at the boundaries between pairs of c chains within a catalytic trimer ( figure below)

The active site of ATCase. 
Some of the crucial active-site residues are shown binding to the inhibitor PALA (shaded gray). Notice that the active site is composed mainly of residues from one c chain, but an adjacent c chain also contributes important
residues (boxed in green).

Each catalytic trimer contributes three active sites to the complete enzyme. Further examination of the ATCase–PALA complex reveals a remarkable change in quaternary structure on binding of PALA. The two catalytic trimers move
12 Å farther apart and rotate approximately 10 degrees about their common threefold axis of symmetry. Moreover, the regulatory dimers rotate approximately 15 degrees to accommodate this motion ( Figure below )

The T-to-R state transition in ATCase. 
Aspartate transcarbamoylase exists in two conformations: a compact, relatively inactive form called the tense (T) state and an expanded form called the relaxed (R) state. Notice that the structure of ATCase changes dramatically in the transition from the T state to the R State. PALA binding stabilizes the R state.

The enzyme literally expands on PALA binding. In essence, ATCase has two distinct quaternary forms: one that predominates in the absence of substrate or substrate analogs and another that predominates when substrates or analogs are bound. We call these forms the T (for tense) state and the R (for relaxed) state, respectively. How can we explain the enzyme’s sigmoidal kinetics in light of the structural observations? Like hemoglobin, the enzyme exists in an equilibrium between the T state and the R state. In the absence of substrate, almost all the enzyme molecules are in the T state because the T state is more stable than the R state. The ratio of the concentration of enzyme in the T state to that in the R state is called the allosteric contstant (L). For most allosteric enzymes, L is on the order of 10^2 to 10^3 .

The enzyme is in the T state when bound to CTP and (2) that a binding site for this nucleotide exists in each regulatory chain in a domain that does not interact with the catalytic subunit (Figure below).

CTP stabilizes the T state. 
The binding of CTP to the regulatory subunit of aspartate transcarbamoylase stabilizes the T state.

Each active site is more than 50 Å from the nearest CTP-binding site. The question naturally arises, How can CTP inhibit the catalytic activity of the enzyme when it does not interact with the catalytic chain? The quaternary structural changes observed on substrate-analog binding suggest a mechanism for inhibition by CTP (Figure below).

The R state and the T state are in equilibrium. 
Even in the absence of any substrate or regulators, aspartate transcarbamoylase exists in equilibrium between the R and the T states. Under these conditions, the T state is favored by a factor of approximately 200.

The binding of the inhibitor CTP to the T state shifts the T-to-R equilibrium in favor of the T state, decreasing net enzyme activity . CTP increases the allosteric coefficient from 200 in its absence to 1250 when all of the regulatory
sites are occupied by CTP. The binding of CTP makes it more difficult for substrate binding to convert the enzyme into the R state. UTP, the immediate precursor to CTP, also regulates ATCase. While unable to inhibit the enzyme alone, UTP synergistically inhibits ATCase in the presence of CTP.

3. Ring closure to form dihydroorotate. 
The third reaction of the pathway is an intramolecular condensation catalyzed by Dihydroorotase to yield dihydroorotate.

Dihydroorotase forms a multifunctional enzyme with carbamoyl phosphate synthetase and aspartate transcarboymalase.
4. Oxidation of dihydroorotate 
Dihydroorotate is irreversibly oxidized to orotate by dihydroorotate dehydrogenase  . The eukaryotic enzyme, which contains FMN and nonheme Fe, is located on the outer surface of the inner mitochondrial membrane, where quinones supply its oxidizing power. The other five enzymes of pyrimidine nucleotide biosynthesis are cytosolic in animal cells. 

Dihydroorotate dehydrogenase The protein encoded by this gene catalyzes the fourth enzymatic step, the ubiquinone-mediated oxidation of dihydroorotate to orotate, in de novo pyrimidine biosynthesis. 

5. Acquisition of the ribose-phosphate moiety
 Orotate reacts with PRPP to yield orotidine-5-monophosphate (OMP) in a reaction catalyzed by Orotate phosphoribosyltransferase. The reaction, which is driven by the hydrolysis of the eliminated PPi, fixes the anomeric form of pyrimidine nucleotides in the beta configuration. Orotate phosphoribosyl transferase also salvages other pyrimidine bases, such as uracil and cytosine, by converting them to their corresponding nucleotides.

Overall structure of Orotate phosphoribosyltransferase ( MtOPRT ) 
Cartoon representation of the dimeric MtOPRT structure. Monomers A and B are coloured in green and pink, respectively; hood domains of protomers A and B are coloured respectively in blue and light blue, the ligand FDC is colored in yellow and the Fe(III) is represented as a light blue sphere; flexible loop of chain B is colored in purple.

6. Decarboxylation to form UMP
The final reaction of the pathway is the decarboxylation of OMP by OMP decarboxylase to form UMP. ODCase enhances the rate (kcat/KM) by a factor of 2 x 10^23 over that of the uncatalyzed reaction, making it the most catalytically proficient enzyme known. Nevertheless, the reaction requires no cofactors to help stabilize its putative carbanion intermediate. Although the mechanism of the ODCase reaction is not fully understood, the removal of OMP’s phosphate group, which is quite distant from the C6 carboxyl group, deceases the reaction rate by a factor of 7 x 10^7, thus providing a striking example of how binding energy can be applied to catalysis (preferential transition state binding ).

OMP decarboxylase is known for being an extraordinarily efficient catalyst capable of accelerating the uncatalyzed reaction rate by an impressive factor of 10^17. To put this in perspective, a reaction that would take 78 million years in the absence of enzyme takes 18 milliseconds when it is enzyme catalyzed. This extreme enzymatic efficiency is especially interesting because OMP decarboxylases uses no cofactor and contains no metal sites or prosthetic groups. The catalysis relies on a handful of charged amino acid residues positioned within the active site of the enzyme. 25

Homodimer Orotidine 5′-monophosphate decarboxylase (ODCase)  
One subunit is colored red and the other blue. Completely conserved residues are emphasized with ball-and-stick representations. The inhibitor, BMP, is drawn in yellow. (A) Viewed through the R/â-barrel. (B) Viewed perpendicular to the view in panel A. . The two subunits are connected by a series of hydrogen bonds between residues His24 and Asp90; Lys73 and Asp76; N79 and Asp200; Arg105 and Ser132, Asp137, and Asp200; His75 and Lys47 and His99; and Asp200 and N79.

A Proficient Enzyme

Reaction scheme of OMP decarboxylation.

In order to make RNA and DNA, a prebiotic earth without this enzyme would have needed to wait 78 million years to yield Uridine monophosphate to make RNA. by natural processes...... 

Enzyme Catalyst:
Enzyme expert Dr Richard Wolfenden, of the University of North Carolina, showed in 1998 that a reaction ‘“absolutely essential” in creating the building blocks of DNA and RNA would take 78 million years in water’, but was speeded up 10^18 times by an enzyme.1 This was orotidine 5′-monophosphate decarboxylase, responsible for de novo synthesis of uridine 5′-phosphate, an essential precursor of RNA and DNA, by decarboxylating orotidine 5′-monophosphate (OMP).
In 2003, Wolfenden found another enzyme exceeded even this vast rate enhancement. A phosphatase, which catalyzes the hydrolysis of phosphate dianions, magnified the reaction rate by thousand times more than even that previous enzyme—10^21 times. That is, the phosphatase allows reactions vital for cell signalling and regulation to take place in a hundredth of a second. Without the enzyme, this essential reaction would take a trillion years—almost a hundred times even the supposed evolutionary age of the universe (about 15 billion years)!3
Wolfenden said,
‘Without catalysts, there would be no life at all, from microbes to humans. It makes you wonder how natural selection operated in such a way as to produce a protein that got off the ground as a primitive catalyst for such an extraordinarily slow reaction.’

Pyrimidine Nucleotide Biosynthesis Is Regulated at ATCase or Carbamoyl Phosphate Synthetase II
In bacteria, the pyrimidine biosynthetic pathway is primarily regulated at Reaction 2, the ATCase reaction (Figure a below).

Regulation of pyrimidine biosynthesis. 
The control networks are shown for (a) E. coli and (b) animals. Red octagons and green circles indicate control points. Feedback inhibition is represented by dashed red arrows, and activation is indicated by dashed green

In E. coli, control is exerted through the allosteric stimulation of ATCase by ATP and its inhibition by CTP In many bacteria, however, UTP is the major ATCase inhibitor. In animals, ATCase is not a regulatory enzyme. Rather, pyrimidine biosynthesis is controlled by the activity of carbamoyl phosphate synthetase II, which is inhibited by UDP and UTP and activated by ATP and PRPP (Figure b above). A second level of control in the mammalian pathway occurs at OMP decarboxylase, for which UMP and to a lesser extent CMP are competitive inhibitors. In all organisms, the rate of OMP production varies with the availability of its precursor, PRPP. Recall that the PRPP level depends on the activity of ribose phosphate pyrophosphokinase.

a Uridine monophosphate (UMP), also known as 5′-uridylic acid (conjugate base uridylate), is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside uridine. UMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase uracil; hence, it is a ribonucleotide monophosphate. As a substituent or radical its name takes the form of the prefix uridylyl-. The deoxy form is abbreviated dUMP. Covalent attachment of UMP (e.g. to a protein such as adenylyltransferase) is called uridylylation (or sometimes uridylation) 24

b In inorganic chemistry, bicarbonate (IUPAC-recommended nomenclature: hydrogencarbonate) is an intermediate form in the deprotonation of carbonic acid. It is a polyatomic anion with the chemical formula HCO−3. 

Bicarbonate HCO3:

c Carbamoyl phosphate is an anion of biochemical significance. In land-dwelling animals, it is an intermediary metabolite in nitrogen disposal through the urea cycleand the synthesis of pyrimidines. 26

5. David Goodsell, Our Molecular Nature, page 26
10. Biochemistry, sixth edition,  Reginald H. Garrett | Charles M. Grisham     page 943
15. Biochemistry, sixth edition,  Reginald H. Garrett | Charles M. Grisham     page 943
16. Origins of Life on the Earth and in the Cosmos,  SECOND EDITION, page 232

28. Lehninger principles of biochemistry, 7th ed. page 887


further readings:
De novo synthesis of pyrimidine nucleotides
Nucleotide Biosynthesis
Pyrimidine Nucleotide Biosynthesis

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3 Purines on Sun May 24, 2015 5:42 pm



A purine is a heterocyclic aromatic organic compound that consists of a pyrimidine ring fused to an imidazole ring. Purines and pyrimidines make up the two groups of nitrogenous bases, including the two groups of nucleotide bases. Two of the four deoxyribonucleotides (deoxyadenosine and deoxyguanosine) and two of the four ribonucleotides (adenosine, or AMP, and guanosine, or GMP), the respective building blocks of DNA and RNA, are purines. In order to form DNA and RNA, both purines and pyrimidines are needed by the cell in approximately equal quantities.  20

De novo synthesis of purines requires a substantial investment of ATP. Purine salvage pathways provide a more economical means of generating purines. Biochemists believe that the enzymes of many metabolic pathways, such as glycolysis and the citric acid cycle, are physically associated with one another. Such associations would increase the efficiency of pathways by facilitating the movement of the product of one enzyme to the active site of the next enzyme in the pathway.

Synthesis of Purine Ribonucleotides

ec00230  Purine metabolism


N1 of purines arises from the amino group of aspartate; C2 and C8 originate from formate; N3 and N9 are contributed by the amide group of glutamine; C4, C5, and N7 are derived from glycine (strongly suggesting that this molecule is wholly incorporated into the purine ring); and C6 comes from HCO-3.

De novo pathway for purine nucleotide synthesis. 
The origins of the atoms in the purine ring are indicated.

When synthesized de novo, purine synthesis begins with simple starting materials such as amino acids and bicarbonate. Unlike the bases of pyrimidines, the purine bases are assembled already attached to the ribose ring. 6
The atoms forming the purine ring are successively added to ribose-5-phosphate; thus, purines are directly synthesized as nucleotide derivatives by assembling the atoms that comprise the purine ring system directly on the ribose. Beginning with ribose-5-P and ending with IMP, the pathway consists of 11 enzymatic steps. 11 

The purine ring system is assembled on ribose-phosphate
De novo purine biosynthesis, like pyrimidine biosynthesis, requires Phosphoribosyl pyrophosphate PRPP but, for purines, PRPP provides the foundation on which the bases are constructed step by step.

Phosphoribosyl-pyrophosphate synthetase (Prs)  catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. PRPP is required for the synthesis of purine and pyrimidine nucleotides, the pyridine nucleotide cofactor NAD(P) and the amino acids histidine and tryptophan. In nucleotide synthesis, PRPP is used both for de novo synthesis and for the salvage pathway, by which bases are metabolized to nucleotides.  Prs is thus a central enzyme in the metabolism of nitrogen-containing compounds. 7

Ribose-phosphate Pyrophosphokinase I 8

The initially synthesized purine derivative is inosine monophosphate (IMP) see below, the nucleotide of the base hypoxanthine. IMP is the precursor of both AMP and GMP.

Inosine monophosphate is synthesized on a pre-existing ribose-phosphate through a complex pathway. The carbon and nitrogen atoms of the purine ring, 5 and 4 respectively, come from multiple sources. The amino acid glycine contributes all its carbon (2) and nitrogen (1) atoms, with additional nitrogen atoms coming from glutamine (2) and aspartic acid (1), and additional carbon atoms coming from formyl groups (2). These are transferred from the coenzyme tetrahydrofolate as 10-formyltetrahydrofolate, along with a carbon atom from bicarbonate (1). Formyl groups build carbon-2 and carbon-8 in the purine ring system, which are the ones acting as bridges between two nitrogen atoms. 5

The metabolic origin of the nine atoms in the purine ring system.

The nine atoms of the purine ring system (Figure above) are contributed by aspartic acid (N-1), glutamine (N-3 and N-9), glycine (C-4, C-5, and N-7), CO2 (C-6), and THF one-carbon derivatives (C-2 and C-8 ). 11
THF is tetrahydrofolate, a coenzyme serving as a one-carbon transfer agent, not only in purine ring synthesis but also in amino acid metabolism  21

Purines are initially formed as ribonucleotides rather than as free bases. Additional studies have demonstrated that such widely divergent organisms as E. coli, yeast, pigeons, and humans have virtually identical pathways for the biosynthesis of purine nucleotides, thereby further demonstrating the biochemical unity of life.

The enzymes of de novo purine synthesis

Phosphoribosyl-pyrophosphate synthetase (Prs) 

1. Ribose-phosphate diphosphokinase
2. amidophosphoribosyl transferase
3. Phosphoribosylglycinamide formyltransferase ( GAR )
4. Phosphoribosylaminoimidazole carboxylase
5. Dihydrofolate reductase
6. AIR synthetase
7. AIR carboxylase
8. SAICAR synthetase
9. adenylosuccinase (adenylosuccinate lyase)
10. AICAR transformylase
11. IMP cyclohydrolase 

The enzymes for Adenine synthesis

1. Adenylosuccinate synthase 
2. adenylosuccinase (adenylosuccinate lyase)

The enzymes for Guanine synthesis

1. IMP dehydrogenase
2. GMP synthase 

Purine Synthesis Yields Inosine Monophosphate
IMP is synthesized in a pathway composed of 11 reactions:

The metabolic pathway for the de novo biosynthesis of IMP
Here the purine residue is built up on a ribose ring in 11 enzyme-catalyzed reactions. The X-ray structures for all the enzymes are shown to the outside of the corresponding reaction arrow. The peptide chains of monomeric enzymes are colored in rainbow order from N-terminus (blue) to C-terminus (red). The oligomeric enzymes, all of which consist of identical polypeptide chains, are viewed along a rotation axis with their various chains differently colored. Bound  ligands are shown in space-filling form with C green, N blue, O red, and P orange.

The de novo pathway for purine synthesis. 
IMP (inosine monophosphate or inosinic acid) serves as a precursor to AMP and GMP. 10

De novo purine biosynthesis. 
(1) Glycine is coupled to the amino group of phosphoribosylamine. 
(2) N10-Formyltetrahydrofolate (THF) transfers a formyl group to the amino group of the glycine residue. 
(3) The inner amide group is phosphorylated and converted into an amidine by the addition of ammonia derived from glutamine. 
(4) An intramolecular coupling reaction forms the five-membered imidazole ring. 
(5) Bicarbonate adds first to the exocyclic amino group and then to a carbon atom of the imidazole ring. 
(6) The imidazole carboxylate is phosphorylated, and the phosphate is displaced by the amino group of aspartate. 
(7) Fumarate is released. 
(8 ) A second formyl group is donated from N10-formyltetrahydrofolate (THF).
(9) Cyclization completes the synthesis of inosinate, a purine nucleotide.

Many of the intermediates in the de novo purine biosynthesis pathway degrade rapidly in water. Their instability in water suggests that the product of one enzyme must be channeled directly to the next enzyme along the pathway. Recent evidence shows that the enzymes do indeed form complexes when purine synthesis is required.

This is remarkable and shows how foreplanning is required to get the end product. There is no natural urge or need for these intermediates to be preserved. 

1. Activation of ribose-5-phosphate. 
The starting material for purine biosynthesis is Ribose 5-phosphate, a product of the pentose phosphate pathway.

That means, the synthesis of ribonucleosides depends on the  pentose phosphate pathway 12

In the first step of purine biosynthesis,  Ribose-phosphate diphosphokinase ( PRPP synthetase).) activates the ribose by reacting it with ATP to form  5-Phosphoribosyl-1-Pyrophosphate (PRPP). This compound is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan. As is expected for an enzyme at such an important biosynthetic crossroads, the activity of ribose-phosphate pyrophosphokinase is precisely regulated.

Ribose-phosphate diphosphokinase

The two major purine nucleoside diphosphates, ADP and GDP, are negative effectors of ribose-5-phosphate pyrophosphokinase.

That rises the question which emerged first : ADP and GDP which are the product of the pathway of which Ribose-phosphate diphosphokinase makes part, or the enzyme per se.

2. Acquisition of purine atom N9. 
In the first reaction unique to purine biosynthesis, Amidophosphoribosyl transferase (ATase) catalyzes the displacement of PRPP’s pyrophosphate group by glutamine’s amide nitrogen.
The reaction occurs with inversion of the  configuration at C1 of PRPP, thereby forming  Beta 5-phosphoribosylamine and establishing the anomeric form of the future nucleotide. The reaction, which is driven to completion by the subsequent hydrolysis of the released PPi, is the pathway’s flux-controlling step.

Amidophosphoribosyl transferase 

This step is the displacement of  Pyrophosphate by ammonia, rather than by a preassembled base, to produce 5-phosphoribosyl-1-amine, with the amine in the beta configuration.

Glutamine phosphoribosyl amidotransferase (ATase) catalyzes this reaction , which is the committed step in purine regulation . This enzyme comprises two domains: the first is homologous to the phosphoribosyltransferases in purine salvage pathways, whereas the second produces ammonia from glutamine by hydrolysis. However, this glutamine-hydrolysis domain is distinct from the domain that performs the same function in carbamoyl phosphate synthetase II . In glutamine phosphoribosyl amidotransferase, a cysteine residue located at the amino terminus facilitates glutamine hydrolysis. To prevent wasteful hydrolysis of either substrate, the amidotransferase assumes the active configuration only on binding of both PRPP and glutamine. As is the case with carbamoyl phosphate synthetase II, the ammonia generated at the glutamine-hydrolysis active site passes through a channel to reach PRPP without being released into solution. 6

The anomeric carbon atom of the substrate PRPP is in the a-configuration; the product is a b-glycoside e (recall that all the biologically important nucleotides are b-glycosides). The N atom of this N-glycoside becomes N-9 of the nine-membered purine ring; it is the first atom added in the construction of this ring. Glutamine phosphoribosyl pyrophosphate amidotransferase is subject to feedback inhibition by GMP, GDP, and GTP as well as AMP, ADP, and ATP. The G series of nucleotides interacts at a guanine-specific allosteric site on the enzyme, whereas the adenine nucleotides act at an A-specific site. The pattern of inhibition by these nucleotides is such that residual
enzyme activity is expressed until sufficient amounts of both adenine and guanine nucleotides are synthesized. Glutamine phosphoribosyl pyrophosphate amidotransferase is also sensitive to inhibition by the glutamine analog azaserine . Azaserine has been used as an antitumor agent because it irreversibly inactivates glutamine-dependent enzymes by reacting with nucleophilic groups at the glutamine-binding site. Two such enzymes are found at steps 2 and 5 of the purine biosynthetic pathway. 15

3.  Acquisition of purine atoms C4, C5, and N7
Glycine’s carboxyl group forms an amide with the amino group of phosphoribosylamine, yielding glycinamide ribotide (GAR). This reaction is reversible, despite its concomitant hydrolysis of ATP to ADP  Pi. It is the only step of the purine biosynthetic pathway in which more than one purine ring atom is acquired. 

This step is carried out by glycinamide ribonucleotide synthetase (GAR synthetase) via its ATP-dependent condensation of the glycine carboxyl group with the amine of 5-phosphoribosyl-b-amine . The reaction proceeds in two stages. First, the glycine carboxyl group is activated via ATP-dependent phosphorylation. Next, an amide bond is formed between the activated carboxyl group of glycine and the b-amine. Glycine contributes C-4, C-5, and N-7 of the purine. 15

4. Acquisition of purine atom C8
GAR’s free alpha amino group is formylated a to yield formylglycinamide ribotide (FGAR). The formyl donor in the reaction is N10-formyltetrahydrofolate (N10- formyl-THF) b , a coenzyme that transfers C1 units . The X-ray structure of the enzyme catalyzing the reaction, GAR transformylase, in complex with GAR and the THF analog 5-deazatetrahydrofolate (5dTHF) was determined by Robert Almassy. Note the proximity of the GAR amino group to N10 of 5dTHF. This supports enzymatic studies suggesting that the GAR transformylase reaction proceeds via the nucleophilic attack of the GAR amine group on the formyl carbon of N10-formyl-THF to yield a tetrahedral intermediate.

Step 4 is the first of two THF-dependent reactions in the purine pathway of eukaryotes. (In E. coli and related organisms, formate, not N 10-formyl-THF, is the source of formyl groups, both here in and in step 10. In prokaryotes, these reactions depend on ATP for formate activation.) GAR transformylase transfers the N 10-formyl group of N10-formyl-THF to the free amino group of GAR to yield a-N-formylglycinamide ribonucleotide (FGAR). Thus, C-8 of the purine is “formyl-ly” introduced. Although all of the atoms of the imidazole portion of the purine ring are now present, the ring is not closed until Reaction 6.

GAR transformylase

N10-formyl tetrahydrofolate (THF)
The active form is tetrahydrofolate (THF). THF is formed through two successive reductions of folate by Dihydrofolate reductase  It is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. 16

Dihydrofolate reductase

5. Acquisition of purine atom N3
The amide amino group of a second glutamine is transferred to the growing purine ring to form formylglycinamidine ribotide (FGAM). This reaction is driven by the coupled hydrolysis of ATP to ADP + Pi.

Step 5 is catalyzed by  FGAR amidotransferase (also known as FGAM synthetase). ATPdependent transfer of the glutamine amido group to the C-4-carbonyl of FGAR yields formylglycinamidine ribonucleotide (FGAM). The imino-N becomes N-3 of the purine.

Phosphoribosylformylglycinamidine syntethase II monomer + 2 ATP (green-red) + 2 Mg ( FGAR amidotransferase

6. Formation of the purine imidazole ring
 The purine imidazole ring is closed in an ATP-requiring intramolecular condensation that yields 5-aminoimidazole ribotide (AIR). The aromatization of the imidazole ring is facilitated by the tautomeric shift of the reactant from its imine to its enamine form.

Step 6 is an ATP-dependent dehydration that leads to formation of the imidazole ring. ATP is used to phosphorylate the oxygen atom of the formyl group, activating it for the ring closure step that follows. Because the product is 5-aminoimidazole ribonucleotide, or AIR, this enzyme is called AIR synthetase. In higher organisms, the enzymatic activities for steps 3, 4, and 6 (GAR synthetase, GAR transformylase, and AIR synthetase) reside on a single multifunctional polypeptide.

7. Acquisition of C6
Purine C6 is introduced as (CO2) in a reaction catalyzed AIR carboxylase by  that yields carboxyaminoimidazole ribotide (CAIR). In yeast, plants, and most prokaryotes (including E. coli ), AIR carboxylase consists of two proteins called PurE and PurK. Although PurE alone can catalyze the carboxylation reaction, its KM for HCO 3 is 110 mM, so the reaction would require an unphysiologically high HCO 3 concentration (100 mM) to proceed. PurK decreases the HCO-3 concentration required for the PurE reaction by 1000-fold but at the expense of ATP hydrolysis.

AIR carboxylase

8. Acquisition of N1
 Purine atom N1 is contributed by aspartate in an amide-forming condensation reaction yielding 5-aminoimidazole-4- (N-succinylocarboxamide) ribotide (SACAIR). This reaction, which
is driven by the hydrolysis of ATP, chemically resembles Reaction 3.

In step 8, the amino-N of aspartate provides N-1 through linkage to the C-6 carboxyl function of CAIR. ATP hydrolysis drives the condensation of Asp with CAIR. The product is N-succinylo -5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR). SAICAR synthetase catalyzes the reaction. The enzymatic activities for steps 7 and 8 reside on a single, bifunctional polypeptide in eukaryotes. 17

Phosphoribosylaminoimidazolesuccinocarboxamide synthase ( SAICAR synthetase )

9. Elimination of fumarate
SACAIR is cleaved with the release of fumarate, yielding 5-aminoimidazole-4-carboxamide ribotide (AICAR). Aspartate’s amino group is transferred to an acceptor through an ATP-driven coupling reaction followed by the elimination of the aspartate carbon skeleton as fumarate.

Step 9 removes the four carbons of Asp as fumarate in a nonhydrolytic cleavage. The product is 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); the enzyme is  adenylosuccinase (adenylosuccinate lyase)  . Adenylosuccinase acts again in that part of the purine pathway leading from IMP to AMP and takes its name from this latter reaction (see following). AICAR is also a by-product of the histidine biosynthetic pathway, but because ATP is the precursor to AICAR in that pathway, no net purine synthesis is achieved.

Adenylosuccinate lyase tetramer + 4 AMP (green) + 4 Cl (orange) + glycerol (, Human

10. Acquisition of C2
 The final purine ring atom is acquired through formylation by N10-formyl-THF, yielding 5-formaminoimidazole-4-carboxamide ribotide (FAICAR). This reaction and Reaction 4 of purine biosynthesis are inhibited indirectly by sulfonamides, structural analogs of the p-aminobenzoic acid constituent of THF.

Step 10 adds the formyl carbon of N10-formyl-THF as the ninth and last atom necessary for forming the purine nucleus. The enzyme is called  AICAR transformylase  ; the products are THF and N-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR).

11. Cyclization to form IMP
 The final reaction in the purine biosynthetic pathway, ring closure to form IMP, occurs through the elimination of water. In contrast to Reaction 6, the cyclization that forms the imidazole ring, this reaction does not require ATP hydrolysis.

Step 11 involves dehydration and ring closure to form the purine nucleotide IMP (inosine-59-monophosphate or inosinic acid); this completes the initial phase of purine biosynthesis. The enzyme is IMP cyclohydrolase (also known as IMP synthase and inosinicase). Unlike step 6, this ring closure does not require ATP. In bacteria and eukaryotes, but not archaea, the enzymatic activities catalyzing steps 10 and 11 (AICAR transformylase and inosinicase) activities reside on a bifunctional polypeptide that dimerizes.

IMP cyclohydrolase

In animals, Reactions 10 and 11 are catalyzed by a bifunctional enzyme, as are Reactions 7 and 8. Reactions 3, 4, and 6 also take place on a single protein. The intermediate products of these multifunctional enzymes are not readily released to the medium but are channeled to the succeeding enzymatic activities of the pathway. As in the reactions catalyzed by the pyruvate dehydrogenase complex, fatty acid synthase, bacterial glutamate synthase
, and tryptophan synthase, channeling in the nucleotide synthetic pathways increases the overall rate of these multistep processes and protects intermediates from degradation by other cellular enzymes.

IMP Is Converted to Adenine and Guanine Ribonucleotides
IMP does not accumulate in the cell but is rapidly converted into Adenosine monophosphate (as AMP) and Guanonsine monophosphate (as GMP) . AMP, which differs from IMP only in the replacement of its 6-keto group by an amino group, is synthesized in a two-reaction pathway ( below figure, on the left ):

Conversion of IMP to AMP or GMP in separate two-reaction pathways. 

In the first reaction to make AMP, aspartate’s amino group is linked to IMP in a reaction powered by the hydrolysis of GTP to GDP + Pi to yield adenylosuccinate. In the second reaction, adenylosuccinate lyase eliminates fumarate from adenylosuccinate to form AMP. The same enzyme catalyzes Reaction 9 of the IMP pathway. Both reactions add a nitrogen with the elimination of fumarate. 

GMP is also synthesized from IMP in a two-reaction pathway (Figure above, on the right ). In the first reaction, IMP is dehydrogenated via the reduction of NAD+ to form xanthosine moophosphate (XMP; the ribonucleotide of the base xanthine). XMP is then xanthine). XMP is then converted to GMP by the transfer of the glutamine amide nitrogen in a reaction driven by the hydrolysis of ATP to AMP + PPi (and subsequently to 2 Pi )

Inonosine monophosphate IMP is the precursor to both AMP (adenosine 5'-monophosphate) and guanine 5'-monophosphate GMP.

The synthesis of AMP and GMP (a) (b) from IMP. 
(a) AMP synthesis: The two reactions of AMP synthesis mimic steps 8 and 9 in the purine pathway leading to IMP. 
(b) GMP synthesis.

These major purine nucleotides are formed via distinct two-step metabolic pathways that diverge from IMP. The branch leading to AMP (adenosine 5'-monophosphate) involves the displacement of the 6-O group of inosine with aspartate (Figure below) in a GTP-dependent reaction f , followed by the nonhydrolytic removal of the four-carbon skeleton of Asp as fumarate; the Asp amino group remains as the 6-amino group of AMP. Adenylosuccinate synthetase and adenylosuccinase are the two enzymes promoting the reaction. 

Adenosine monophosphate (AMP) synthesis

Adenylosuccinate synthase  is an enzyme that plays an important role in purine biosynthesis, by catalysing the guanosine triphosphate (GTP)-dependent conversion of inosine monophosphate (IMP) and aspartic acid to guanosine diphosphate (GDP), phosphate and N(6)-(1,2-dicarboxyethyl)-AMP. 

Recall that adenylosuccinase also acted at step 9 in the pathway from ribose-5-phosphate to IMP. Fumarate production provides a connection between purine synthesis and the citric acid cycle.

Adenylosuccinate lyase tetramer + 4 AMP (green) + 4 Cl (orange) + glycerol (, Human  converts adenylosuccinate to AMP. The same enzyme catalyzes Reaction 9 of the IMP pathway. 

Guanosine monophosphate (GMP) synthesis
GMP is also synthesized from IMP in a two-reaction pathway. In the first reaction, IMP is dehydrogenated via the reduction of NAD+ to form xanthosine monophosphate (XMP; the ribonucleotide of the base xanthine). XMP is then converted to GMP by the transfer of the glutamine amide nitrogen in a reaction driven by the hydrolysis of ATP to AMP+ PPi (and subsequently to 2 Pi ). 

The formation of GMP from IMP requires oxidation at C-2 of the purine ring, followed by a glutamine-dependent amidotransferase reaction that replaces the oxygen on C-2 with an amino group to yield 2-amino,6-oxy purine nucleoside monophosphate, or as this compound is commonly known, guanosine monophosphate (Figure above). The enzymes in the GMP branch are IMP dehydrogenase and GMP synthetase. Note that, starting from ribose-5-phosphate, 8 ATP equivalents are consumed in the synthesis of AMP and 9 in the synthesis of GMP.

IMP dehydrogenase It catalyzes the rate-limiting reaction of de novo Guanine (GTP) biosynthesis

GMP synthase  In the de novo synthesis of purine nucleotides, IMP is the branch point metabolite at which point the pathway diverges to the synthesis of either guanine or adenine nucleotides. In the guanine nucleotide pathway, there are 2 enzymes involved in converting IMP to GMP, namely IMP dehydrogenase (IMPD1), which catalyzes the oxidation of IMP to XMP, and GMP synthetase, which catalyzes the amination of XMP to GMP

ATP synthesis
The products of de novo purine biosynthesis are the nucleoside monophosphates AMP and GMP. These nucleotides are converted by successive phosphorylation reactions into their metabolically prominent triphosphate forms, ATP and GTP. The first phosphorylation, to give the nucleoside diphosphate forms, is carried out by two basespecific, ATP-dependent kinases, Adenylate kinase and Guanylate kinase

What came first, ATP or the enzymes that use ATP, to make ATP ? 

ATP drives proteins that make AMP. ATP drives enzymes that make ADP. ATP drives enzymes that make ATP. ATP drives proteins that make AMP. ATP drives enzymes that make ADP. ATP drives enzymes that make ATP.  ====>>> endless loop. 

The Adenine triphosphate (ATP) molecule as energy source is required to drive the enzymes/protein machines that make the adenine nucleic base and adenosine monophosphate AMP, used in DNA, one of the four genetic nucleotides "letters" to write the Genetic Code, and then, using these nucleotides as starting material, further molecular machines attach other two phosphates and produce adenine triphosphates (ATP) - he very own molecule which is used as energy source to drive the whole process.. What came first: the enzymes to make ATP, or ATP to make the enzymes that make ATP?

Nucleoside Diphosphates and Triphosphates Are Synthesized by the Phosphorylation of Nucleoside Monophosphates.
In order to participate in nucleic acid synthesis, nucleoside monophosphates must first be converted to the corresponding nucleoside triphosphates.  First, nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific  nucleoside monophosphate kinases .  The two substrates of this enzyme are ATP and nucleoside monophosphate, whereas its two products are ADP and nucleoside diphosphate.  This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with a phosphate group as acceptor

Purine Nucleotide Biosynthesis Is Regulated at Several Steps
The pathways synthesizing IMP, ATP, and GTP are individually regulated in most cells so as to control the total amounts of purine nucleotides available for nucleic acid synthesis, as well as the relative amounts of ATP and GTP.

Control of the purine biosynthesis pathway. 
Red octagons and green circles indicate control points. Feedback inhibition is indicated by dashed red arrows, and feedforward activation is represented by a dashed green arrow.

Purines Can Be Salvaged
In most cells, the turnover of nucleic acids, particularly some types of RNA, releases adenine, guanine, and hypoxanthine. These free purines are reconverted to their corresponding nucleotides through salvage pathways. In contrast to the de novo purine nucleotide synthetic pathway, which is virtually identical in all cells, salvage pathways are diverse in character and distribution.

Adenine and Guanine
Both adenine and guanine are derived from the nucleotide inosine monophosphate (IMP), which in turn is synthesized from a pre-existing ribose phosphate through a complex pathway using atoms from the amino acids glycine, glutamine, and aspartic acid, as well as the coenzyme tetrahydrofolate.

Prebiotic synthesis of Purines
The only alternative to these biochemical processes would be, that the basic building blocks were readily available on a prebiotic earth. Glycine for instance is a indispensable substrate for purine nucleotide synthesis, and so - DNA - in cells. It requires at least 5 biosynthetic steps and the respective enzymes to be synthesized. In a prebiotic earth, the only alternative would have been that glycine came from comets.

Comet contains glycine, key part of recipe for life 3
May 27, 2016
An important amino acid called glycine has been detected in a comet for the first time, supporting the theory that these cosmic bodies delivered the ingredients for life on Earth, researchers said Friday.
In addition to the simple amino acid glycine, the instrument also found phosphorus. The two are key components of DNA and cell membranes. "Demonstrating that comets are reservoirs of primitive material in the Solar System, and vessels that could have transported these vital ingredients to Earth, is one of the key goals of the Rosetta mission, and we are delighted with this result."

Panspermia, not a viable explanation for the OOL

Chemistry happens, and interesting molecules form in space; so what?  It’s not going to help the believers in naturalistic origin of life.  So they found glycine, the simplest and only non-chiral amino acid.  The biologists told the astronomers to look for life’s building blocks in space, because they were having such a hard time producing them on Earth.  They would need megatons of amino acids and nucleic acid bases to rain down on the Earth for any hope of getting successful concentrations, but then the precious cargo would be subject to rapid degradation by water, oxygen, UV light, and harmful cross-reactions.  Even then, they would be mixtures of left and right handed forms, with no desire nor power to organize themselves into astronomers who could invent weird science like this.
Following the unresolved issues of nucleotide biosynthesis


In biochemistry, the addition of a formyl functional group is termed formylation. A formyl functional group consists of a carbonyl bonded to hydrogen. When attached to an R group, a formyl group is called an aldehyde. 3

b 10-Formyltetrahydrofolate (10-CHO-THF) is a form of tetrahydrofolate that acts as a donor of formyl groups in anabolism. In these reactions, 10-CHO-THF is used as a substrate in formyltransferase reactions. This is important in purine biosynthesis, where 10-CHO-THF is a substrate for phosphoribosylaminoimidazolecarboxamide formyltransferase, as well as in the formylation of the methionyl initiator tRNA (fMet-tRNA), when 10-CHO-THF is a substrate for methionyl-tRNA formyltransferase 4 

c  In chemistry, a pyrophosphate is a phosphorus oxyanion. Compounds such as salts and esters are also called pyrophosphates. The group is also called diphosphate or dipolyphosphate, although this should not be confused with phosphates. 9

d In biochemistry, a kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. 13

e In chemistry, a glycoside /ˈɡlaɪkəsaɪd/ is a molecule in which a sugar is bound to another functional group via a glycosidic bond. Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. These can be activated by enzyme hydrolysis,[1] which causes the sugar part to be broken off, making the chemical available for use. 14

f  Guanosine-5'-triphosphate (GTP) is a purine nucleoside triphosphate. It is one of the building blocks needed for the synthesis of RNA during the transcription process. Its structure is similar to that of the guanine nucleobase, the only difference being that nucleotides like GTP have a ribose sugar and three phosphates, with the nucleobase attached to the 1' and the triphosphate moiety attached to the 5' carbons of the ribose. 
In the cell, GTP is synthesised through many processes including:
as a byproduct of the Succinyl-CoA to Succinate conversion catalysed by the Succinyl-CoA synthetase enzyme as part of the Krebs cycle
through exchanges of phosphate groups from ATP molecules by the Nucleoside-diphosphate kinase, an enzyme tasked with maintaining an equilibrium between the concentrations of different nucleoside triphosphates. 19

6. Biochemistry 8th ed. Styer, page 748
10. Biochemistry, Garrett , 6th edition , page 929
11. Biochemistry, Garrett , 6th edition , page 928
15. Biochemistry 6th ed. Garrett, page 930
17. Biochemistry 6th ed. Garrett, page 932

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4 Formation of Deoxyribonucleotides on Wed May 27, 2015 1:27 am


Formation of Deoxyribonucleotides

DNA (Deoxyribonucleic acid) is the core of life in Earth, every known living organism is using DNA as their genetic backbone. DNA is so precious and vital to eukaryotes that its kept packaged in cell nucleus, it's being copied but never removed because it never leaves the safety of nucleus. DNA directs all cell activity by delegating it to RNA. RNA (Ribonucleic acid ) have varied sort of biological roles in coding, decoding, regulation, and expression of genes. RNA carries messages out of the cell nucleus to cytoplasm. 7

The structure of RNA nucleotides is very similar to that of DNA nucleotides, with the main difference being that the ribose sugar backbone in RNA has a hydroxyl (-OH) group that DNA does not. This gives DNA its name: DNA stands for deoxyribonucleic acid. Another minor difference is that DNA uses the base thymine (T) in place of uracil (U). Despite great structural similarities, DNA and RNA play very different roles from one another in modern cells. 8

The synthesis of deoxyribonucleotides, the precursors of DNA are formed by the reduction of ribonucleotides; specifically, the 2'-hydroxyl group a on the ribose moiety is replaced by a hydrogen atom. The substrates are ribonucleoside diphosphates, and the ultimate reductant is NADPH. The enzyme ribonucleotide reductase is responsible for the reduction reaction for all four ribonucleotides. The ribonucleotide reductases of different organisms are a remarkably diverse set of enzymes. Yet detailed studies have revealed that they have a common reaction mechanism, and their three-dimensional structural features indicate that these enzymes are homologous. 2

DNA differs chemically from RNA in two major respects:

(1) Its nucleotides contain 2'-deoxyribose residues rather than ribose residues, and
(2) it contains the base thymine (5-methyluracil) rather than uracil. In this section, we consider the biosynthesis of these DNA components.

In ribonucleotides, the pentose is ribose, while in deoxyribonucleotides (or just deoxynucleotides), the sugar is 2'-deoxyribose (i.e., the carbon at position 2' lacks a hydroxyl group a ). 

                  Ribose                                            Deoxyribose

From the figure above we can see that the principal difference between the two molecules is the presence of OH in ribose (2' tail) and absence in deoxyribose. There is a difference in one Oxygen atom as the name stands de-oxy ribose. Both Ribose and deoxyribose have an Oxygen(O) atom and a Hydrogen (H) atom (an OH group) at their 3' sites. The OH groups are very reactive in nature, so the 3' OH tail is required for phosphodiester bonds to form between nucleotides in both ribose and deoxyribose atoms.

DNA is such an important molecule so it must be protected from decomposition and further reactions. The absence of one Oxygen is the key to extend DNA's longevity. When the 2' Oxygen is absent in deoxyribose, the sugar molecule is less likely to get involved in chemical reactions( the aggressive nature of Oxygen in chemical reactions are famous). So by removing the Oxygen from deoxyribose molecule, DNA avoids being broken down. In an RNA's point of view the Oxygen is helpful, unlike DNA, RNA is a short-term tool used by the cell to send messages and manufacture proteins as a part of gene expression. Simply speaking mRNA (Messenger RNA) has the duties of turning genes ON and OFF, when a gene needed to be put ON mRNA is made and to keep it OFF the mRNA is removed. So the OH group in 2' is used to decompose the RNA quickly thereby making those affected genes in OFF state.

The ribose sugar is placed in RNA for easily decomposing it and DNA uses deoxyribose sugar for longevity.

G.F.Joyce wrote in a 2002 Nature review article:
The primary advantage of DNA over RNA as a genetic material is the greater chemical stability of DNA, allowing much larger genomes based on DNA. 9  To expand, RNA is unsuitable for large genomes because the 2'-OH of ribose (obviously absent from the 2'-dexoyribose of DNA) renders the phosphodiester bond susceptible to alkaline hydrolysis .

RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a hydroxyl group at the 2’ position. This feature makes RNA chemically unstable compared to DNA, which does not have this 2’ OH group and thus is not susceptible to base-catalyzed hydrolysis 10  This will occur slowly at pH 7.6, but at a rate calculated to be sufficient to degrade a 1000 nucleotide RNA in about 70 days.  if this 2’-Hydroxyl (-OH) group is removed from the ribose sugar than the rate of such base-catalyzed hydrolysis is decreased by approximately 100 fold under extreme conditions. Thus, the presence of 2’-Hydroxyl (-OH) group on every nucleotide of RNA makes it labile and easily degradable.

The presence of Thymine at the place of Uracil in DNA.
The replacement of RNA as the repository of genetic information by its more stable cousin, DNA, provides a more reliable way of transmitting information. This explains why DNA uses thymine (T) as one of its four informational bases, whereas RNA uses uracil (U) in its place. The problem is that cytosine (C), one of the two other bases, can easily turn into U, through a simple reaction called deamination. This takes place spontaneously dozens of times a day in each of your cells but is easily corrected by cellular machinery because, in DNA, U is meaningless. However, in RNA such a change would be significant – the cell would not be able to tell the difference between a U that was supposed to be there and needed to be acted upon, and a U that was a spontaneous mutation from C and needed to be corrected. This does not cause your cells any difficulty, because most RNA is so transient that it does not have time to mutate – in the case of messenger RNA it is copied from DNA immediately before being used. Thymine is much more stable and does not spontaneously change so easily. The adoption of DNA as the genetic material, with its built-in error-correction mechanism in the shape of the two complementary strands in the double helix, and the use of thymine in the sequence, provided a more reliable information store and slowed the rate of potentially damaging mutations. 12

The only structural difference between Thymine and Uracil is the presence of methyl group in Thymine. This methyl group facilitates the repair of damaged DNA, providing an additional selective advantage. Cytosine in DNA undergoes spontaneous deamination at a perceptible rate to form Uracil. For example, under typical cellular conditions, deamination of Cytosine to Uracil (in DNA) occurs in about every 107  Cytidine residues in 24 hours, which means 100 spontaneous events per day. The deamination of Cytosine is potentially mutagenic because Uracil pairs with Adenine and this would lead to a decrease in G≡C base pairs and increase in A=U base pairs in DNA of all cells. Over the time period, the Cytosine deamination could completely eliminate G≡C base pairs. But, this mutation is prevented by a repair system that recognizes Uracil as foreign in DNA and removes it.

Thus, the methyl group on thymine is a tag that distinguishes thymine from deaminated cytosine. But, if DNA normally contains Uracil recognition would be more difficult and unpaired Uracil would lead to permanent sequence changes as they were paired with Adenine during replication. So, we can say that Thymine is used in place of Uracil in DNA to enhance the fidelity of the genetic message. In contrast, RNA is not repaired and so Uracil is used in RNA because it is a less expensive building block. 11

Chemical structures of nucleotides.
(a) A 5'-ribonucleotide and
(b) a 3'-deoxynucleotide. The purine or pyrimidine base is linked to C1' of the pentose and at least one phosphate (red) is also attached. A nucleoside consists only of a base and a pentose.

When the phosphate group is absent, the compound is known as a nucleoside. A 5'-nucleotide can, therefore, be called a nucleoside-5'-phosphate. Nucleotides most commonly contain one to three phosphate groups at the C5' position and are called nucleoside monophosphates, diphosphates, and triphosphates. The structures, names, and abbreviations of the common bases, nucleosides, and nucleotides are given in Table below. 

Ribonucleotides are components of RNA (ribonucleic acid), whereas deoxynucleotides are components of DNA (deoxyribonucleic acid). Adenine, guanine, and cytosine occur in both ribonucleotides and deoxynucleotides (accounting for six of the eight common nucleotides), but uracil primarily occurs in ribonucleotides and thymine occurs in deoxynucleotides. Free nucleotides, which are anionic, are almost always associated with the counterion Mg2+ in cells.

Ribonucleotide reductases

- Overall description
- function
- structure
- classes
- mechanism and reaction
- biosynthesis
- regulation
- repair
- origin and evolution

In all living cells, a ribonucleotide reductase (RNR) provides the deoxyribonucleoside triphosphates (dNTPs) for DNA replication and repair. A single enzyme replaces the OH group at C2′ of the ribose moiety of the four common ribonucleotides with hydrogen. The reaction occurs with either ribonucleoside di- or triphosphates as substrates. Different organisms employ widely different forms of three different classes of the enzyme and, in some cases, contain more than one enzyme. All classes of RNR share two exceptional features:

1. The polypeptide chain of the active enzyme harbors a free radical amino acid residue that participates in the catalytic process; the mechanism for radical generation sets the classes apart
2. The specificity toward the four ribonucleotides is tightly controlled by allosteric effects that are remarkably similar for the three classes. 64 

DNA components are synthesised from their corresponding ribonucleotides by the reduction of the C2' position. The remarkable enzymes that do this are named Ribonucleotide reductases (RNR). RNRs are mechanistically fascinating because of their free radical chemistry, unusual metallocofactors and complex regulatory mechanisms. 58 It is a very important enzyme for all living organisms 52  The iron-dependent enzyme is essential for DNA synthesis. It is one of the most essential enzymes of life 32  It is remarkable that RNR uses some of the most potent metals in redox chemistry 47 All RNRs use radical chemistry to catalyze this challenging reaction. Despite diverse means of radical generation and storage, the three classes of RNR are unified by the subsequent channelling of the radical into a thiyl radical species g in the structurally conserved active site 48

It is crucial that these dNTP pools are carefully balanced since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors g and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. 50 

RNR's provide an essential link between the RNA and DNA world. 

Ribonucleotide reduction is the only pathway for de novo synthesis of deoxyribonucleotides in extant organisms. This chemically demanding reaction, which proceeds via a carbon-centred free radical. The mechanism has been deemed unlikely to be catalyzed by a ribozyme, creating an enigma regarding how the building blocks for DNA were synthesized at the transition from RNA to DNA-encoded genomes. 33

That brings us to the classic chicken and egg, catch22 situation.  RNR enzymes are required to make DNA. DNA is however required to make RNR enzymes. What came first ??  We can conclude with high certainty that this enzyme buries any RNA world fantasies and any possibility of transition from  RNA to DNA world scenarios.

Biosynthesis DNA is made from RNA. The deoxynucleotides are made from nucleotides with ribonucleotide reductases (RNR's), producing uracil-DNA or u-DNA. The uracil is then converted to thymine by adding a methyl group, making thymine-DNA or t-DNA, the kind that is actually used.

The reduction pathway performed by RNR's is almost certainly as old as the ( supposed ) divergence of archaebacteria, eubacteria, and eukaryotes, as it is found in all modern organisms studied to date 14 A first prerequisite for DNA synthesis is the balanced supply of the different deoxyribonucleotide triphosphates (dNTPs). The only biochemical pathway for de novo dNTP synthesis is the reaction catalyzed through the enzyme ribonucleotide reductase (RNR), which converts the four ribonucleotides triphosphates (NTPs) into their corresponding dNTPs through the reduction of the C2′-OH bond.

Ribonucleotide Reductase (RNRs) Convert Ribonucleotides to Deoxyribonucleotides (dNTPs)
RNRs are essential enzymes to sustain life in all free-living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors for DNA synthesis and repair.  Ribonucleotide reductase (RNR) is the enzymatic machine that maintains this pool of DNA precursors together with the so-called salvage pathways. RNR catalyzes the reduction of the four main ribonucleotides to their corresponding deoxyribonucleotide diphosphates or deoxyribonucleotide triphosphates depending on the three classes of RNR. RNR's activity is highly transcriptionally regulated and cell phase dependent. To avoid imbalanced levels of dNTPs and the increased mutation rates that are the inevitable consequences of this RNRs are tightly controlled through transcriptional and allosteric regulation, subcellular compartmentalization and small protein inhibitors Allosteric regulation of RNR's affects both substrate specificity and overall activity. The s-site binds dNTPs and determines which nucleotide will be reduced at the active site to ensure balanced levels of the four deoxyribonucleotides dNTPs in the cell.

This is a goal-oriented process. Since it had to emerge prior when life began, and there was no need of survival of the fittest, the origin of this selection is best explained by an intelligent agency with distant goals in mind. 

The presence of 2-deoxyribose as the sugar in DNA nucleotides rather than the ribose found in RNA comes about through reduction of the ribose sugar on a ribonucleotide substrate by Ribonucleotide Reductases. Some aerobic bacteria and all anaerobic microorganisms studied carry out this reduction at the ribonucleoside tri-phosphate (rNTP) level. In all other organisms studied, however, the substrates for ribonucleotide reductase are the ribonucleoside 5′ -di-phosphates (rNDPs). 63

One of the essential processes for sustaining life in all organisms is the availability of a balanced pool of DNA building blocks for processes such as cell division and DNA damage repair. Ribonucleotide reductase (RNR) is the enzymatic machine that maintains this pool of DNA precursors together with the so-called salvage pathways. The RNR system represents both the initial and the rate-limiting step in the DNA-synthesis, as this process is allosterically regulated, especially in higher organisms. RNRs activity is highly transcriptionally regulated and cell phase dependent. 27 

Ribonucleotide reductases (RNRs) transform RNA building blocks to DNA building blocks by catalyzing the substitution of the 2OHgroup of a ribonucleotide with a hydrogen by a mechanism involving protein radicals. 22 Deoxyribonucleotides are synthesized from their corresponding ribonucleotides by the reduction of their C2' position rather than by their de novo synthesis from deoxyribose-containing precursors.

In a ribonucleotide or a deoxyribonucleotide, one or more phosphate groups are bonded to atom C3' or atom C5' of the pentose to form a 3'-nucleotide or a 5'-nucleotide, respectively

The deoxyribonucleotides have only one metabolic purpose: to serve as precursors for DNA synthesis. In most organisms, ribonucleoside diphosphates (NDPs) are the substrates for deoxyribonucleotide formation. Reduction at the 2'-position of the ribose ring in NDPs produces 2'-deoxy forms of these nucleotides (Figure above). This reaction involves replacement of the 2'-OH by a hydride ion (H;-) b and is catalyzed by an enzyme known as ribonucleotide reductase. Enzymatic ribonucleotide reduction involves a free radical mechanism. Enzymes that catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides are named ribonucleotide reductases (RNRs)

The first RNR activity was observed in the year 1950 by a Swedish researcher Peter Reichard and coworkers, where they observed the conversion of ribonucleotides to deoxyribonucleotides.  Seven decades after its discovery, RNR is still a popular field to study in the scientific community. Perhaps, it is no exaggeration to say that RNR is the most interesting enzyme to study. 51 Although it has been almost seven decades since the isolation of nucleotide reductase, RNR continues to surprise after all these years. 53

The transcription of the two RNR genes occurs exclusively during S-phase in eukaryotic cells. Due to the long half-life of the R1 subunit, its concentration level is practically constant throughout the cell cycle and always in excess relative to the R2 subunit. Hence, cell cycle-dependent activity of RNR is mainly regulated by synthesis and degradation of R2 subunit. The tyrosyl radical is generated in the smaller R2 subunit (also denoted and the radical is shuttled to the active site of the large R1 subunit for turnover. This process triggers formation of a thiyl radical, which is required for substrate activation and reduction. In class Ia RNR, electrons are provided by redox active cysteines of thioredoxin (Trx) enzymes and glutaredoxin enzymes.  

Class Ib with Mn-cluster requires participation of the NrdI and NrdH proteins for generation of the tyrosyl radical in R2 and the reduction of the catalytic site in R1, respectively. Class Ib also differs from class Ia RNRs because the ATP-cone domain in the large subunit is absent, thus class Ib has less allosteric regulation. Class Ic was recently discovered in mammalian parasites such as Chlamydia trachomatis. Here the class Ia and class Ib tyrosyl radical
site is replaced by a phenylalanine residue. Notably, a comparable coordination environment of the iron-ions is also present in class Ia protein Epstein-Barr Virus R2, based on the protein sequence. 62

RNR structure
RNR uses radical chemistry to catalyze the reduction of each NTP. How the enzyme generates this radical, the type of cofactor and metal required, the three-dimensional structure of this enzyme complex and the dependence of oxygen are all characteristics that are considered when classifying RNRs.

The X-ray structures of the R1 catalytic component and the R2 di-iron component of the class I RNR from E. coli were determined . The catalytic subunit was found to be a novel 10-stranded α/β-barrel with a loop that hosts the thiyl radical protruding into its center.  Despite a lack of sequence similarity with other ribonucleotide reductases, all ribonucleotide reductases would have similar catalytic subunits, reflecting their similar catalytic strategies. The structure of the catalytic subunit of the class III enzyme revealed the characteristic 10-stranded α/β-barrel with a central loop bearing the thiyl radical precursor. Many features of the structure reported are remarkably similar to the structures of the catalytic subunits of the class I and class III enzymes, even though there is <10% sequence homology among them. The similarities and contrasts with the enzymes of other classes have much to tell us about all ribonucleotide reductases.

The three classes of RNR enzymes have the same catalytic activity, but different amino acid sequences to reach the same result. Science has no good explanations for the divergence.

The three classes of ribonucleotide reductases 55

The three different classes of RNR
Models of the active class I α2β2 complex (based on the crystal structures of the R1 and R2 proteins); class III (1HK8), dimeric class II (PDB 30O0) and monomeric class II. The monomeric class II enzymes have maintained within one polypeptide chain the minimal structural elements required for substrate specificity regulation. Substrates and allosteric effectors are shown as space-filling models. The adenosylcobalamin cofactors in the class II enzymes are shown as sticks. The box at the upper right shows a homology model of a β-monomer for the class III enzymes.

The  3 classes of RNR 
Three classes of RNRs have been identified; they all share a structurally homologous α subunit which binds the NDP(NTP) substrates, houses the two cysteines that provide the reducing equivalents for dNDP(dNTP) formation, and a third cysteine that must be oxidized transiently to a thiyl radical to initiate the reduction process. 
Currently, three different RNR classes have been described (I, II, and III), and class I is further subdivided into Ia, Ib, and Ic. All three RNR classes share a common three-dimensional protein structure at the catalytic subunit and a highly conserved α/β barrel structure in the active site of the enzyme. In addition, the two potential allosteric centers (specificity and activity) are highly conserved among the different RNR classes, although in class Ib, and some class II RNRs activity allosteric site is absent. 30  Reduction of the four different nucleotides (ATP/CTP/GTP/TTP) occurs at a single active site in each polypeptide chain, therefore the tight regulation of dNTP levels is important for each dividing cell. Unbalanced dNTP levels could lead to increased mutation rates .

The reduction of ribonucleotides to deoxyribonucleotides by RNR.
Three different RNR classes (I, II, and III) have been described for this enzyme family. RNR is important for evolution, as this enzyme played an important role during the transition from an RNA to a DNA world. RNR enzymes catalyze the reduction of the ribose C2′-OH to C2′-H.

The significant differences between RNRs exist notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes. 31

Crystal structures of the catalytic subunit of the three RNR classes 
(A) class I E. coli R1
(B) class II Lactobacillus leichmannii NrdJ  and
(C) class III bacteriophage T4 
All the catalytic subunits have a similar ten-stranded alpha/beta barrel with a cysteine residue (indicated by a yellow sphere) placed in the middle of the active site

There are three classes of RNRs, which differ in their prosthetic groups, although they all replace the 2'-OH group of ribose with H via a free-radical mechanism.

The schematic model of three RNR classes system showing the reduction of ribonucleotides to their corresponding deoxyribonucleotides (see insert on top right). 
The catalytic site is in the larger subunit in class I NrdE/R1E/NrdA/R1, while the tyrosyl radical site is located in the smaller subunit NrdF/R2F/NrdB/R2. Thioredoxin/glutaredoxin/NrdH reduces a disulphide bond in NrdA/R1E/NrdJ, while formate reduces anaerobically NrdD. The NrdI protein is needed for the formation of tyrosyl radical in Mn-Mn containing R2F. Class Ia RNR has only di-Fe site, while class Ic has no tyrosyl radical and Fe–Mn site. Class II has a vitamin B12 (cobalamin) radical initiator and Class III uses SAM, an iron-sulfur cluster and glycyl-radical as radical initiators. 28 

Table above summarizes the organisms where RNRs have been found to date, together with some of their characteristics (metal cofactors, redox partners and allosteric sites). Overview of the various classes of ribonucleotide reductase and their division in three main classes (I, II and III).

Class I
Class I reductases are aerobic enzymes present in all higher organisms and certain microorganisms. They consist of two homodimers named R1 and R2. Protein R1 contains the binding sites for both substrates and allosteric effectors and redox active cysteines for the reduction of ribonucleotides. Electrons are provided by NADPH, through electron transfer chains (thioredoxin or thioredoxin reductase ). Protein R2 contains a non-heme diiron center in which the two ferric ions are oxo-bridged and an adjacent tyrosyl radical. It is proposed that the function of the radical is to abstract, through a long-range electron transfer pathway, one electron from a cysteine residue of R1 and thus to generate a thiyl radical in the proximity of the substrate. 55

Class I is found in higher organisms and in some prokaryotes and viruses. Class I has been further divided into three subclasses, Ia–c, due to diversities in polypeptide sequence homologies, overall structural regulation behaviors, metal cofactors (Fe–Fe, Fe–Mn, or Mn–Mn), and natural source of reducing equivalents. Class Ia RNRs occurs in a wide range of organisms: eukaryotes, prokaryotes, viruses and bacteriophages, but class Ib has only been found in prokaryotes similar to those of the anaerobic class III. All class I three subclasses contain two non-identical subunits, R1 and R2. 

The class I RNRs require a second subunit β which houses the essential metallocofactor and is required for thiyl radical formation in α in an oxidation that occurs over a 35 Å distance in an unprecedented process in biology. The class I RNRs have been sub-classified based on their metal composition. The class Ia RNRs are found in eukaryotes (ex., human and Saccharomyces cerevisiae) and a few prokaryotes (ex., Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium)). The class Ib RNRs are found in most prokaryotes (ex. E. coli, Corynebacterium ammoniagenes, Bacillus subtilis, Streptococcus sanguinis, Bacillus cereus, and Bacillus anthracis). A few prokaryotes possess both Ia and Ib RNRs. . The Ia RNRs utilize a diferric-tyrosyl (FeIII 2-Y•) radical cofactor and the Ib RNRs are able to use a diferric or dimanganic-tyrosyl radical (MnIII 2-Y•) cofactor. 61 

Class I RNRs are found in both prokaryotes and eukaryotes with few exceptions. They are characterized by a tyrosyl radical that is stabilized by an oxo-bridged binuclear Fe(III) complex and requires oxygen for its generation. 57  . The enzyme is a holoenzyme, being the R1 protein composed by two identical monomers (761 residues per monomer) each one lodging one active site (for reduction of purines and pyrimidines), constituted by five conserved residues, Cys439, Cys225, Cys462, Glu441 and Asn437, and three independent allosteric sites named s-site (specificity site), a-site (adenine specific site) and h-site (hexamerization site) that control the activity of the enzyme. The other dimer b2, named R2 has 375 residues in each monomer, each one containing a stable neutral tyrosyl free radical at position 122, coupled to a binuclear iron (Fe2O2) cluster required for generation and stabilization of the radical.

Class I RNR is a tetramer composed of large (RNR1) and small (RNR2) subunits. Class I RNR is iron-dependent and produces tyrosyl radical. Thimidine triphosphate (TTP) is an effector in the reaction.
Class I RNRs consist two subgroups (Ia, Ib, and Ic) which differ only slightly in primary structure

Class I is divided into subclasses
Ia (tyrosyl-radical and di-iron-oxygen cluster) 
Ib (tyrosyl-radical and di-manganese-oxygen cluster) 
Ic (an iron-manganese cluster) 

(a) Structures and (b) metallocofactors of the class I RNR β2 subunits. 
(a) The class Ia E. coli NrdB, the class Ib E. coli NrdF, and the class Ic Chlamydia trachomatis NrdB. FeIII and MnII ions are shown as brown and purple spheres. (b) The metals and protein residues involved in metal binding are shown in cartoon form. The metal site closest to Tyr or Phe is termed site 1 (Mn1, Fe1) and the other is site 2 (Mn2, Fe2). Because the coordination modes of the Asp and Glu residues are dependent on the oxidation state of the cluster, no metal-ligand bonds are drawn. The structure of the class Ia FeIIIFeIII-Y• cofactor has been established. The class Ib MnIIIMnIII-Y• has been crystallized; however, owing to the sensitivity of manganese to photoreduction, the oxidation states of the manganese ions and the number and identity of bridging ligands are not clear. The MnIVFeIII cofactor of the class Ic cluster has not been crystallographically characterized, and in the structure shown, the proposed placements of manganese and iron are based on extended X-ray absorption fine structure (EXAFS) data.

Structures of the reduced (left) and oxidized (right) metallocofactors of the class I ribonucleotide reductases.
Solvent molecules are shown as red spheres, and iron and manganese ions are brown and purple spheres.  E. coli FeIIFeII-NrdB (1PIY), E. coli FeIIIFeIII-NrdB (1MXR), E. coli MnIIMnII-NrdF (3N37), Corynebacterium ammoniagenes MnIIIMnIII-NrdF (3MJO), E. coli FeIIFeII-NrdF (3N38), Salmonella enterica serovar Typhimurium FeIIIFeIII-NrdF (2R2F), and C. trachomatis FeIIIFeIII-NrdB (1SYY).

Both subgroups are common in that they contain two different dimeric subunits (R1 and R2) and require oxygen in order to form a stable radical. Class Ic RNRs are the most recently discovered. Evidence also suggests its existence in archaea and eubacteria. The sequence of class Ic RNRs shows that residues in the PCET pathway and active site for nucleotide reductase are similar between the three subgroups. 16

All eukaryotes from yeast to humans contain class Ia enzymes. Class Ib occurs in a large spectrum of eubacteria. Both classes require oxygen to function, and active enzymes are not found in strict anaerobes.

Class II
Class II RNR reduces ribonucleotide triphosphates using coenzyme B12.
Class II enzymes are found in many microorganisms, both aerobes and anaerobes. They consist of a single polypeptide, either as a monomer or as a dimer, and use adenosylcobalamin (AdoCbl) to generate a cysteinyl radical.
Adenosylcobalamin (AdoCbl), which is also known as cobamamide and dibencozide, is, along with methylcobalamin (MeCbl), one of the active forms of vitamin B12. 60
As in class I enzymes the substrates are reduced by redox active cysteines and electrons are provided by enzymatically reduced thioredoxin or glutaredoxin. 55

Class II RNRs form thiyl radicals with the help of adenosylcobalamin – which fulfills the role of the R2 subunit as a radical generator – and utilize thioredoxin or glutaredoxin as electron donors. Therefore, class II RNRs are made up of only one subunit and present as monomers or dimmers and neither require nor are inhibited by the presence of oxygen. 

Class II reductases are microbial enzymes that occur in both aerobic and anaerobic organisms.

Class III
Class III RNR generate glycine radical using S-adenosyl methionine and Fe-S center. 4
Class III enzymes are found in some anaerobically growing facultative anaerobes and are extremely sensitive to oxygen.. The large subunit a2 contains binding sites specific for allosteric effectors (nucleoside triphosphates) that regulate the activity and specificity of the enzyme. It is likely that it also contains the binding site for the substrates because it harbors, in its active form, a glycyl radical absolutely required for activity. Whether this radical serves to generate a thiyl radical as in class I and I1 enzymes is still speculative. The small subunit p2  contains an iron-sulfur center. This center is a f4Fe-4SJ cluster which brings the two p chains together, but more work is necessary to confirm this point. The radical enzyme is competent for the reduction of ribonucleotides, at the expense of the reducing equivalents of formate, as shown by the stoichiometric production of C02 during turnover. So, at variance with the two other classes of RNRs, the class III enzyme employs a low molecular weight compound as the external reductant. 

Class III RNRs, like Class I RNRs, are made up of two dimeric protein subunits (NrdG and NrdD); however, unlike in Class I RNRs which require R2 continuously to generate radicals, the small NrdG is only required during the activation of NrdD. The mechanism of Class III RNRs uses formate as an electron donor and generates an oxygen-sensitive glycyl radical, thus rendering the enzymes inactive in the presence of oxygen.

Class III reductases depend on anaerobiosis and are found in both strict anaerobic and facultative anaerobic organisms.

The three metal RNR Co-factors
Class I of Ribonucleotide reductases occurs in aerobically thriving organisms including humans uses oxygen activated by a dinuclear iron center to convert a tyrosine residue into a radical. 
Class II is the coenzyme B12-dependent reductase
Class III contains an extremely oxygen-sensitive glycyl radical, which is generated with the aid of S-adenosylmethionine (SAM). 

The three well-characterized metallocofactors for RNRs and PFL, and their generation of an active site S•. 
Each of the amino acid radicals is part of the protein. The Y• is part of the R2 subunit in class I RNRs
The cofactor for class II RNRs is AdoCbl. CH2Ad is the axial ligand, the corrin is represented by the rectangle and the bottom face axial ligand, dimethylbenzimidazole, by the vertical filled-in rectangle. 
The G• is part of the α2 subunit of class III RNRs and PFL. The S• is part of R1 of class I RNRs, α2 of class III RNRs and the α subunit of class II RNRs. 

All three types, however, use a thiyl radical at the active site and act by an almost identical mechanism.

Different classes of RNR's have intriguing sequence “motifs” involving cysteines that appear to be important for the catalysis (in Escherichia coli, Cys-439, the radical site, and Cys-225 and Cys-462, which delivers two electrons and a proton). These motifs offer tantalizing suggestions that all RNRs are related by common ancestry but underwent divergent evolution so massive that only traces of evidence for homology remain in the sequences themselves. These motifs are inadequate to provide a statistically significant case for homology, however, and motifs are notoriously inadequate for confirming homology in general. 14

To claim common ancestry, in this case, is an ad-hoc assertion. Truth said, science is unable to infer a reasonable scenario out of the evidence, and all it can do, is resort to made-up stories, which bear no credibility. The best and straightforward explanation is that a creator made RNR's, and equipped each of them with different ways to perform the same function.

Metallo cofactors structures (FeIII or MnIII) found in class Ia, class Ib and Ic RNR
(A) E. coli  (class Ia) with both metal centers occupied by iron
(B) mouse (class Ia) with both metal centers occupied by iron, 
(C) S. typhimurium  (class Ib) with both metal centers occupied by iron, 
(D) C. ammoniagenes (class Ib) with both metal centers occupied by manganese, 
(E) C. trachomatis  (class Ic) with both metal centers occupied by iron and 
(F) C. trachomatis (coordinates was obtained from Martin Högbom) with one metal center occupied by iron and the other by manganese.

a hydroxy or hydroxyl group is the entity with the formula OH. It contains oxygen bonded to hydrogen. In organic chemistry, alcohol and carboxylic acids contain hydroxy groups. The anion [OH], called hydroxide, consists of a hydroxy group. 1

Representation of an organic hydroxy group, where R represents a hydrocarbon or other organic moiety, the red and grey spheres represent oxygen and hydrogen atoms respectively, and the rod-like connections between these, covalent chemical bonds.

b In chemistry, a hydride is the anion of hydrogen, H, or, more commonly, it is a compound in which one or more hydrogen centers have nucleophilic, reducing or basic properties. In compounds that are regarded as hydrides, the hydrogen atom is bonded to a more electropositive element or group. Compounds containing hydrogen bonded to metals or metalloid may also be referred to as hydrides. 5
An ion is an atom or molecule that has a non-zero net electrical charge (its total number of electrons is not equal to its total number of protons). A cation is a positively-charged ion, while an anion is negatively charged. Because of their opposite electric charges, cations and anions attract each other and readily form ionic compounds. 6 Since the electric charge on a proton is equal in magnitude to the charge on an electron, the net electric charge on an ion is equal to the number of protons in the ion minus the number of electrons.
An anion, , meaning "up", is an ion with more electrons than protons, giving it a net negative charge (since electrons are negatively charged and protons are positively charged).
cation, meaning "down", is an ion with fewer electrons than protons, giving it a positive charge.

c Adenosylcobalamin (AdoCbl), which is also known as cobamamide and dibencozide, is, along with methylcobalamin (MeCbl), one of the active forms of vitamin B12 18

d In chemistry, a thiyl radical has the formula RS, sometimes written RS• to emphasize that they are free radicals. R is typically an alkyl or aryl substituent. 19

e Cysteine biosynthesis 

f Nicotinamide adenine dinucleotide phosphate ( NADPH ) , abbreviated NADP+ is a cofactor used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent. 41  Cells require a constant supply of NADPH for reductive reactions vital to biosynthetic purposes. Much of this requirement is met by a glucose-based metabolic sequence called the pentose phosphate pathway 45  . In addition to providing NADPH for biosynthetic processes, this pathway produces ribose-5-phosphate, which is essential for nucleic acid synthesis.  It is a cytoplasmic branch of glycolysis that provides both NADPH and ribose for nucleotide synthesis. 44 

g In biochemistry, an effector molecule is usually a small molecule that selectively binds to a protein and regulates its biological activity. In this manner, effector molecules act as ligands that can increase or decrease enzyme activity, gene expression, or cell signaling. Effector molecules can also directly regulate the activity of some mRNA molecules (riboswitches). 59

2. Biochemistry, 8th ed. Styer, page  753
3. Biochemistry, 6th ed. Garrett, page 946
12. Life's greatest secrete, page 178
13. Encyclopedia of life ciences, page 5063
17. Enzyme-Catalyzed Electron and Radical Transfer, page 358
44. Biochemistry, 6th ed. Garrett, page 780
47. The Origin and Evolution of Ribonucleotide Reduction

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5 RNR Mechanism and reaction on Fri May 29, 2015 3:27 pm


RNR Mechanism  and reaction
All ribonucleotide reduction proceeds via controlled free-radical-based chemistry, in which a free radical amino acid residue of an RNR generates a substrate radical by abstracting a hydrogen atom from C3′ of the substrate, to facilitate the leaving of the OH group on the vicinal C2′ (Figure below, a ). A thiyl group of a cysteine residue performs this function. Two additional redox-active cysteine residues then provide the reducing equivalents for the subsequent reduction at C2′. This general mechanism has strong experimental support for class I and II enzymes. The three-dimensional structure of the catalytic site of the E. coli class I enzyme beautifully fits this mechanism(Figure below, b). For class III RNR, the evidence for a similar mechanism is indirect. 59

(a) Free radical mechanism for the reduction of a ribonucleotide. A protein radical (thiyl for class I and II) abstracts the hydrogen from C3′ of the ribotide, thereby transferring the radical function to the substrate. Subsequently, C2′ of the activated substrate is reduced by redox-active cysteine residues, and the abstracted hydrogen is restored to C3′, with regeneration of the protein radical. 
(b) Three-dimensional structure of the large protein of E. coli with a substrate modeled into the catalytic site. The suggested thiyl of Cys439 is located on the b-face of the ribose ring in close vicinity to C3′, whereas the redox-active Cys462 and Cys225 are strategically located on the opposite a face, ready for reduction of C2′.

Class I enzymes, found in E. coli and virtually all eukaryotes, are Fe-dependent and generate the required free radical on a specific tyrosyl side chain. 3 

How ribonucleotide reductase (RNR) generates all four of these building blocks (of DNA) and maintains the correct balance among them has been a long-standing puzzle. Unlike RNR, most enzymes specialize in converting just one type of molecule to another, "Ribonucleotide reductase is very unusual. I've been fascinated with this question of how it actually works and how this enzyme's active site can be molded into four different shapes." They found that the enzyme's active site -- the region that binds the substrate -- changes shape depending on which effector molecule is bound to a distant site on the enzyme. It's exquisitely designed so that if you have the wrong substrate in there, you can't close up the active site. "It's a really elegant set of movements that allow for this kind of molecular screening process." 35 

The enzyme system for dNDP formation consists of four proteins, two of which constitute the ribonucleotide reductase proper, an enzyme of the a2 b2 type. The other two proteins, thioredoxin and thioredoxin reductase, function in the delivery of reducing equivalents. The two proteins of ribonucleotide reductase are designated R1  and R2, and each is a homodimer in the holoenzyme (Figure below).

E. coli ribonucleotide reductase.

Ribbon diagram of the hRRM1 dimer.
Chains A and B are colored yellow and cyan, respectively. The four-helix ATP-binding cones of both subunits are shown in red. TTP (green), GDP (orange), and ATP/dATP (magenta) bound at the S-, C- and A-sites, respectively, are represented as transparent surfaces. 52

On the left: 
The four-helix cone with ATP-bound. 2Fo-Fc electron density for ATP (carbon, oxygen and nitrogen atoms are colored yellow, red and blue, respectively) contoured at 1σ is shown in green wire mesh.
On the right: 

The four-helix cone with dATP-bound. 2Fo-Fc electron density for dATP (carbon, oxygen and nitrogen atoms are colored black, red and blue, respectively) contoured at 1σ is shown in blue wire mesh.

The R1 homodimer carries two types of regulatory sites in addition to the catalytic site (the active site). Substrates (ADP, CDP, GDP, and UDP) bind at the catalytic site. One regulatory site—the substrate specificity site—binds ATP, dATP, dGTP, or dTTP, and which of these nucleotides is bound there determines which nucleoside diphosphate is bound at the catalytic site. The other regulatory site, the overall activity site, binds either the activator ATP or the negative effector dATP; the nucleotide bound here determines whether the enzyme is active or inactive. Activity depends also on residues Cys439, Cys225, and Cys462 in R1. The R2 homodimer is the source of the free radical that initiates the ribonucleotide reductase reaction. Its 2 Fe atoms generate this free radical on R2 Tyr122, which in turn generates a thiyl free radical (Cys-S) on Cys439. Cys439-S initiates ribonucleotide reduction by abstracting the 3'-H from the ribose ring of the nucleoside diphosphate substrate (Figure below) and forming a free radical on C-3'. Subsequent dehydration forms the deoxyribonucleotide product.

Modern protein ribonucleotide reductases. 
A simpli¢ed scheme summarising the chemistry of ribonucleotide reduction for each of the three classes of ribonucleotide reductase. In the ¢rst step, a radical is generated at some distance from the active site. In class I and III, radical generation and catalysis occur on different subunits. The class I reductases produce a stable tyrosine radical with the help of an ironoxygen centre, and this is transferred to the active site, 30^40 Aî away. In classes II and III, a nucleotide cofactor (adenosylcobalamin (AdoCbl) and AdoMet, respectively) is cleaved to generate a deoxyadenosyl radical (AdoCHb 2 ), which subsequently forms a stable glycine radical in the class III reductase. In the next step the radical in all three classes is transferred to an active site cysteine, forming a thiyl radical. This is then transferred to the 3P position of the ribose, and two cysteines act as reductants in classes I and II, while formate is the reductant in the class III reaction. Once the 2P-OH has been reduced to 2P-H, with regeneration of the thiyl radical, this is transferred back to the tyrosine in class I, to the glycine in class III, while in class II AdoCbl is regenerated. In the class III reaction, AdoMet is consumed in generating the glycine radical, but like the tyrosine radical in the class I reaction, the glycine radical is stable and can presumably support several enzymatic turn-over cycles.

The free radical mechanism of ribonucleotide reduction. 
Ha designates the C-39 hydrogen, and Hb, the C-29 hydrogen atom.

A variety of experimental evidence led  to formulate the following catalytic mechanism for E. coli ribonucleotide reductase:
1. The free radical on Tyr 122β is transferred to Cys 439α (really the transfer of an electron from Cys 439α to Tyr 122β) via a mechanism to form a thiyl radical. The thiyl radical abstracts the H atom from C3′ of the substrate NDP in the reaction’s rate-determining step, thereby transferring the radical to C3′.
2. Base-catalyzed abstraction of a proton from O3′ by Glu 441α, accompanied by the migration of the radical to C2′, induces the C2′ OH group to abstract a proton from Cys 462α and leave as H2O.
3. The resulting radical intermediate is reduced by the enzyme’s redox-active sulfhydryl pair (Cys 462α and Cys 225α) to yield a disulfide bond and a 2′-deoxynucleotide with the radical on C3′.
4. The radical migrates to Cys 429α in a reversal of Step 1 to re-form the thiyl radical and yield the enzyme’s dNDP product.
5. The dNDP product is released and replaced by a new NDP substrate. The protein thioredoxin in its reduced form (see below) carries out a disulfide exchange reaction with the redox-active disulfide group on the α subunit,
yielding oxidized thioredoxin and the enzyme in its initial state, thus completing the catalytic cycle.

The Tyr 122β radical, which is located ∼10 Å below the surface of the protein, is remarkably stable: It has a half-life of 4 days at 4°C. The Tyr 122β radical is >35 Å from Cys 439α, too far for the direct transfer of an electron. Rather, the protein mediates this electron transfer via the side chains of a series of redox-active aromatic residues (Tyr and Trp) bridging the α and β subunits. In each step of this process, an electron transfer is accompanied by a proton transfer, ending in the abstraction of an electron and a proton from Cys 439.

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure below:

Proposed mechanism for ribonucleotide reductase.
In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the α subunit. The active-site radical (—X•) is on the β subunit and in E. coli is probably a thiyl radical of Cys439

The reaction performed by RNR's  has been shown to occur in a surprisingly complicated sequence of steps 47 The active site is composed by a set of five amino acids namely, Cys225, Asn437, Cys439, Glu441 and Cys462 and the radical is allocated at Cy439.  

In the first step, the 3´-H atom is abstracted by the radical sulfur of Cys439; 
Second is the transfer of a proton from the 3´-OH group to Glu441, with concomitant protonation of the 2´-OH group by Cys225 and elimination of a water molecule; the 
third step corresponds to the abstraction of an H atom from the thiol group of Cys462 by carbon C2´ - directly or with mediation of the closer Cys225 -; the resulting disulfide radical anion is subsequently reduced to a standard disulfide bond and a proton is transferred from Glu441 to the 3´-O atom, which replaces the charge in the glutamate residue and the spin density in the C3´ carbon; the 
The fourth and final step consists of the donation of the H atom by Cys439 back to that carbon; thus, the 2´-deoxyribonucleotide is formed and the essential tyrosyl radical is regenerated. In the end of this process the disulfide bridge is oxidized by Thioredoxin and the enzyme is ready for a new reduction cycle. 

Structural insight into catalysis

Ribonucleotide reductase active site.

The β strands of the 10-stranded α,β barrels of 
(a) the E. coli class I RNR R1 subunit, 
(b) the T4 phage class III RNR α subunit and 
(c) the PFL α subunit, showing common active site structures. 
The 10 strands are labeled A through J based on the nomenclature for the class I RNR. The residues in ball-and-stick rendition are known to be involved in catalysis in each of the enzymes (see text for further details). In the class I RNR, GDP is placed in the active site. In the class III RNR, G580 was replaced with an alanine for crystallization purposes. The dashed line between residues 543 and 573 implies structural disorder. The N and C termini of the proteins are indicated. 54   the components harboring the active site region in each class are related. 55

Class I E. coli ribonucleotide reductase
Several structures of E. coli R1 have confirmed a number of important features of catalysis. The three cysteines essential for nucleotide reduction are located within 6–7 Å of one another, in an unusual 10-stranded (labeled A–J) α,β-barrel structure (Figure a above).  The barrel is composed of two sets of five contiguous parallel strands forming two sheets that are antiparallel with respect to one another. A finger loop (residues 427–457) projects through the center of the barrel, connecting βE and βF. At the tip of the loop is the C439 residue, which initiates radical-dependent nucleotide reduction. The C225 and C462 residues, which are oxidized concomitant with substrate reduction, are found on two adjacent antiparallel strands (βA and βF). The C-terminal domain of R1 forms a protective surface over part of the active site and provides part of a putative electron transfer pathway involving C439, Y731 and Y730 to R2.

Class II L. leichmannii ribonucleotide reductase
L. leichmannii class II RNR utilizes AdoCbl as a cofactor, is monomeric and is allosterically regulated. The AdoCbl abstracts the hydrogen atom from the cysteine (C408) directly, generating cob(II)alamin located approximately 6 Å from the S•. The remarkable similarities in the chemistry of the class I E. coli and class II L. leichmannii RNRs suggested that the active site of the L. leichmannii RNR would be very similar to that of the E. coli enzyme. As in the case of the class I and III RNRs and PFL, the active site is an α,β barrel with a finger loop containing C408. The similarities in the active site structures of the three classes of RNRs, and the similarities in their chemistry together provide strong support for the hypothesis that these proteins are related. 

Class III T4 phage ribonucleotide reductase Class III RNRs 
They have a quaternary structure. The E. coli class III enzyme was originally proposed to have an α2β2 structure. β2 can catalytically generate a G• on α2.The structure of α2 reveals a 10-stranded α,β barrel with the same connectivity as that observed in R1 from class I RNRs. Together they suggest that C290 is the precursor to the S•. C79 of T4 phage class III RNR is in a similar location to C225 of R1 from class I RNR. A C462 equivalent, essential in class I and II RNRs, is not present. Formate is proposed to act as the direct reductant, replacing the disulfide chemistry.  

Plumbing diagrams for 
(a) the E. coli class I RNR R1 subunit, 
(b) the T4 phage class III RNR α subunit and 
(c) the PFL α subunit. The α,β barrels are in the center, in a similar orientation to that found in Figure 3. The diagram for the class III enzyme was generated using Rasmol with the PDB restrictions removed. Adjacent to the plumbing diagram, the amino acids in each of the β strands are enumerated.

Deoxyribonucleotides are made from ribonucleoside diphosphates. These are ADP, CDP, GDP, and UDP and the enzyme responsible for this catalyzes the conversion of all of them. In the reaction NDP, the N stands for any of the bases A, G, C, or U. NDP is converted to the deoxy equivalent of those (dGDP, dADP, dCDP, and dUDP) by this enzyme. This involves loss of a water. If we look at CDP, what happens in the catalysis by this enzyme is the production of dCDP and what has happened is the hydroxyl at position 2 on the sugar has been removed. That’s the deoxy part of the deoxyribonucleotides that oxygen is gone. Now, ribonucleotide reductase gets changed in the process of that. It starts out in a reduced state and it ends up being oxidized. We know when an enzyme changes it has to be changed back because if don’t change back, we get to a dead end where we have a dead enzyme and we can’t have that enzyme being dead because it’s necessary for making other deoxyribonucleotides. Well, it’s converted back to the reduced form by action of a molecule called thioredoxin. Thioredoxin can reduce and oxidize ribonucleotide reductase and in the process, it becomes oxidized. Well, you start thinking do we have to recycle that? The answer is yes we do. To replenish the thioredoxin in a reduced form, electrons are donated ultimately from any NADPH. They get to the thioredoxin by several steps, the thioredoxin is regenerated so that they can regenerate the ribonucleotide reductase. Now ribonucleotide reductase is a fascinating enzyme in terms of the number of things that it balances and does for being a relatively small protein.

It has 2 subunits, a large subunit and a small subunit. The large subunit is called R1 and it has 2 allosteric sites. The allosteric sites, of course, are sites that bind other molecules and those molecules affect the enzyme’s activity and the active site is at least partly in the large subunits kind of shared between the large and small subunits. The small subunit’s primary function is that it has a tyrosine amino acid within it that gets radicalized and that radicalization of the tyrosine is necessary for the reaction mechanism that produces the deoxyribonucleotides. RNR, ribonucleotide reductase, as it's called, controls the balance of all the deoxyribonucleotides and it does it with complex allosteric controls. It’s a complicated process. Ribonucleotide reductase starts out in a reduced form, and by the end of the reaction, it’s in an oxidized form. The ribonucleotide reductase in the oxidized form has to be cooxidized back to the reduced form and that happens as a result of action of a thioredoxin. Well, how this does these all thing occur? Remember that I said also that the process starts with the tyrosyl radical into small subunit of the ribonucleotide reductase. That radical actually pulls proton off of the ring of carbon #3 on the ribose.  So tyrosine is pulling that hydrogen off and in the process of doing that it takes the electron with it leaving behind a radical that’s on the ring. That creates some instability on the ribose and that instability on the ribose causes the hydroxyl position to pull a hydrogen off of the sulfhydryl of ribonucleotide reductase. You’ll notice that that creates an H2O at that point as shown in the 3rd molecule. Well we can see this process happening and that H2O is unstable.

That H2O is lost, so the loss of the H2O results in now a ribose that has had the radical transferred to another position, that below that of position 2. Well, that radical is very much seeking a hydrogen, and we can see that that hydrogen is lost here from the other sulfur of the sulfhydryl on the ribonucleotide reductase to stabilize the overall sugar. At this point, we have now made the deoxyribose sugar. The radical has to be regenerated and that radical is regenerated by fixing the other part of the radical on the deoxyribose sugar that happens here, and as a result, the ribonucleotide reductase enzyme is completely regenerated into the radical state and simply has to be reduced by thioredoxin. 54
Starting at 2.23:
So in general I have written it as NDP that is nucleotide triphosphate because this is a nucleotide here base is there sugar is there and two phosphates attached to the sugar that is why this is an diphosphate molecule now to synthesize DNA you need deoxynucleotide triphosphate that means ribose sugar that is present here which is a pentose sugar this has to be in deoxy form and how to make this ribose into deoxyribose for that you need to remove this oxygen atom present in this hydroxyl group and it is the second carbon the second carbon of ribose as what is a hidroxyl  here if you remove this oxygen atom so that means you are creating a deoxyribose sugar so that is as simple as that and  how this will be done in our body for that you need this enzyme called ribonucleotide reductase what is ribonucleotide reductase does is it is going to use a low molecular weight protein called thioredoxin ( thio = sulphur groups. It stores its hydrogens in cysteine groups that form a sulphur bridge once oxidized ) in its reduced state and you can see thioredoxin in two thiol groups, that is  SH and SH now those two protons present in these two thiol groups they will be removed by ribonucleotide reductase enzyme so basically thioredoxin SH and SH that is two protons will be going to ribonucleotide reductase and then ribonucleotide reductase is going to insert those two protons and take this oxygen out and put it as water molecules so this is how thioredoxin getting into the reaction and you come out of the reaction as oxidized thioredoxin where you can see there is a disulphide created overall what ribonucleotide reductase has done here is it is going to take this oxygen out take two protons from here and release water molecules and at that time what you're done is basically we have created a deoxy form of ribose as you can see second carbon do not have a  hydroxil group now and this molecule is a deoxynucleotide triphosphates so like this ADP can be converted to dDP that is resonant diphosphate can be converted to the oxygen index phosphate CDP can be converted to dcdp UDP can be converted to dUDP and the GDP can be converted to dDGP so this is what is the job of ribonucleotide reductase so what happened here is thioredoxin is oxidized here now the oxidized thyoredoxine it has to be converted back into its reduced form because we need to maintain sufficient quantities of thioredoxin in the cell so that ribonucleotide reductase continue to do its function so how this is made possible now the oxidized thioredoxin will be converted back into its reduced form by an enzyme called thioredoxin reductase. now this thioredoxin reductase we do this through protons coming from nadp h plus h plus and oxidize NADH + H+ into nadp+ while it is reducing thioredoxin back into its reduced form and that is the job of thioredoxin reductase enzyme there is one more molecule that can be used here by ribonucleotide reductase and that is glutaredoxin instead of thioredoxin so glutaredoxin can also be used and glutaredoxin also as 2SH groups and at the end of the reaction you get oxidized glutaredoxin similar to thioredoxin.

All classes of RNRs catalyze the reduction of ribonucleotides to deoxyribonucleotides and require a metallo-cofactor to generate a thiyl radical that initiates this process.
A, all RNRs have a structurally homologous α subunit (red) where nucleotide reduction occurs and is initiated by the thiyl radical. RNRs are classified based on the metallo-cofactors (blue) that oxidize the active site cysteine into the transient thiyl radical. This oxidation in the class I RNRs requires a second subunit, β (circled in black), designated NrdB for the Ia and NrdF for the Ib proteins. The oxidation uses AdoCbl for the class II RNR (NrdJ) and a glycyl radical for the class III RNR (NrdD). The glycyl radical is generated by an activating enzyme NrdG (black) that requires S-adenosylmethionine (SAM) and a [4Fe4S]+ cluster. In the class I and II RNRs, deoxynucleotide formation is accompanied by oxidation of two cysteines to a disulfide and consequently requires Trx, TrxR, and NADPH for multiple turnovers. NrdH-redoxin can function as a Trx in reduction of class Ib RNRs. In the class III RNRs, formate is the reductant. 
B, O2 is required to assemble the active MnIII2-Y• and FeIII2-Y• cofactors of NrdF (class Ib RNR), which then act catalytically with NrdE to produce deoxynucleotides. The glycyl radical cofactor of the class III RNR (NrdD) is inactivated by O2. C, structure of the AdoCbl cofactor used by the class II RNRs. Ado, adenosyl; AdoCbl, adenosylcobalamin; Cbi (in gray), cobinamide; DMB, dimethylbenzimidazole. 49

A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme’s overall activity by binding ATP (activator) or dATP (inhibitor). 50

Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides by direct reduction at the 2′-carbon atom of the D-ribose to form the 2′-deoxy derivative. De novo synthesis of deoxyribonucleotides is a chemically demanding reaction, which proceeds via a carbon-centred free radical.  The mechanism has been deemed unlikely to be catalyzed by a ribozyme, creating an enigma regarding how the building blocks for DNA were synthesized at the transition from RNA to DNA-encoded genomes.

For example, adenosine diphosphate(ADP) is reduced to 2′-deoxyadenosine diphosphate (dADP), and GDP is reduced to dGDPThis reaction is somewhat unusual in that the reduction occurs at a nonactivated carbon; no closely analogous chemical reactions are known. The reduction of the D-ribose portion of a ribonucleoside diphosphate to 2′-deoxy-D-ribose requires a pair of hydrogen atoms, which are ultimately donated by NADPH via an intermediate hydrogen-carrying protein, thioredoxin. This ubiquitous protein serves a similar redox function in photosynthesis

Thioredoxins act as electron donors to ribonucleotide reductase 39 
Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in deoxyribonucleotide synthesis essential for DNA replication and repair. RNR in S phase mammalian cells comprises a weak cytosolic complex of the catalytic R1 protein containing redox active cysteine residues and the R2 protein harboring the tyrosine free radical. Each enzyme turnover generates a disulfide in the active site of R1, which is reduced by C-terminally located shuttle dithiols leaving a disulfide to be reduced. Electrons for reduction come ultimately from NADPH via thioredoxin reductase and thioredoxin (Trx) or glutathione reductase, glutathione, and glutaredoxin (Grx).

Thioredoxin has pairs of —SH groups that carry hydrogen atoms from NADPH to the ribonucleoside diphosphate. Its oxidized (disulfide) form is reduced by NADPH in a reaction catalyzed by thioredoxin reductase (Figure below), and reduced thioredoxin is then used by ribonucleotide reductase to reduce the nucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs).

A second source of reducing equivalents for ribonucleotide reductase is glutathione (GSH). Glutathione serves as the reductant for a protein closely related to thioredoxin, glutaredoxin, which then transfers the reducing power to ribonucleotide reductase (Figure above). Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in biochemical transformations, once thought to be rare in biological systems.

The enzyme in E. coli and most eukaryotes is an α2β2 dimer, with two catalytic subunits, α2, and two radical-generation subunits, β2 (Figure below). Each catalytic subunit contains two kinds of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the catalytic (α2) and radical-generation (β2) subunits. At each active site, an α subunit contributes two sulfhydryl groups required for activity, and the β2 subunits contribute a stable tyrosyl radical. The β2 subunits also have a binuclear iron (Fe3+) cofactor that helps generate and stabilize the Tyr122 radical (Figure below). The tyrosyl radical is too far from the active site to interact directly with the site, but several aromatic residues form a long-range radical-transfer pathway to the active site (Figure c below).  In E. coli, the sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above.

Ribonucleotide reductase. 
(a) A schematic diagram of the subunit structures. Each catalytic subunit (α; also called R1) contains the two regulatory sites described in Figure below,  and two Cys residues central to the reaction mechanism. The radical-generation subunits (β; also called R2) each contain a critical Tyr122 residue and binuclear iron center. 
(b) The likely structure of α2β2. 
(c) The likely path of radical formation from the initial Tyr122 in a β subunit to the active-site Cys439, which is used in the mechanism shown in Figure 22-43. Several aromatic amino acid residues participate in long-range transfer of the radical from the point of its formation at Tyr122 to the active site, where the nucleotide substrate is bound. 

Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. 
The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site, the substrate-specificity site (right). The diagram indicates inhibition or stimulation of enzyme activity with the four different substrates. The pathway from dUDP to dTTP is described below

Oligomerization of ribonucleotide reductase induced by the allosteric inhibitor dATP. 
At high concentrations of dATP (50 μM), ringshaped α4β4 structures form. In this conformation, the residues in the radicalforming path are exposed to the solvent, blocking the radical reaction and inhibiting the enzyme. The oligomerization is reversed at lower dATP concentrations.

Thioredoxin Reduces Ribonucleotide Reductase
NADPH is the ultimate source of reducing equivalents for ribonucleotide reduction, but the immediate source is reduced thioredoxin, a small protein with reactive Cyssulfhydryl groups situated near one another in the sequence Cys-Gly-Pro-Cys. These Cys residues are able to undergo reversible oxidation–reduction between (OSOSO) and (OSH HSO) and, in their reduced form, serve as primary electron donors to regenerate the reactive OSH pair of the ribonucleotide reductase active site. In turn, the sulfhydryls of thioredoxin must be restored to the (OSH HSO) state for another catalytic cycle. Thioredoxin reductase, an a2-type enzyme composed of  flavoprotein subunits, mediates the NADPH-dependent reduction of thioredoxin (Figure below).

The (OSOSO)/(OSH HSO) oxidation–reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH

Ribbon representation of the dimer of rat TrxR.
The two subunits are shown in light or dark colors, respectively. Red, FAD binding domain; yellow, NADP binding domain; blue, interface domain. Bound FAD (red) and NADP (orange) are shown as ball-and-stick models. 36

Thioredoxin functions in a number of metabolic roles besides deoxyribonucleotide synthesis, the common denominator of which is reversible sulfide; sulfhydryl transitions. The substrates for ribonucleotide reductase are CDP, UDP, GDP, and ADP, and the corresponding products are dCDP, dUDP, dGDP, and dADP. Because CDP is not an intermediate in pyrimidine nucleotide synthesis, it must arise by dephosphorylation of CTP, for instance, via nucleoside diphosphate kinase action. Although uridine nucleotides do not occur in DNA, UDP is a substrate. The formation of dUDP is justified because it is a precursor to dTTP, a necessary substrate for DNA synthesis.

The final step in the ribonucleotide reductase catalytic cycle is reduction of the enzyme’s newly formed disulfide bond to re-form its redox-active sulfhydryl pair. One of the enzyme’s physiological reducing agents is thioredoxin, a ubiquitous monomeric protein with a pair of neighboring Cys residues (and which also participates in regulating the Calvin cycle). Thioredoxin reduces oxidized ribonucleotide reductase via disulfide interchange.

Thioredoxin plays a crucial role in a wide number of physiological processes, in special the reduction of nucleotides to deoxyriboucleotides. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). Thioredoxin Reductase (TrxR) is a ubiquitous homodimeric flavoenzyme whose physiological role is the transfer of reducing equivalents from NADPH to thioredoxin 42 Thioredoxin (Trx) and thioredoxin reductase (TrxR) plus NADPH, comprising the thioredoxin system functions in DNA synthesis.  The amino acid sequence of E. coli Trx1 with 108 residues was determined in 1968  demonstrating the universally conserved active site -Cys-Gly-Pro-Cys-. 46 The most general description of the Trx system is its role as a protein disulfide reductase (Figure below)

NADPH is the ultimate source of reducing equivalents for ribonucleotide reduction, but the immediate source is reduced thioredoxin, a small (12-kD) protein with reactive Cyssulfhydryl groups situated near one another in the sequence Cys-Gly-Pro-Cys.

The mechanism of the so-called Class I RNRs contain an Fe or Mn prosthetic group which occur in most eukaryotes and aerobic prokaryotes. Class I RNRs reduce ribonucleoside diphosphates (NDPs) to the corresponding deoxyribonucleoside diphosphates (dNDPs). E. coli ribonucleotide reductase is mainly present in vitro as a heterotetramer that can be decomposed into two catalytically inactive homodimers, R12 and R22 (Figure a; opposite).

Class I ribonucleotide reductase from E. coli.
(a) Schematic diagram of the quaternary structure. The enzyme consists of two identical pairs of subunits, R12 and R22. Each R2 subunit contains a binuclear Fe(III) complex that generates a phenoxy radical at Tyr 122. The R1 subunits each contain three different allosteric effector sites and five catalytically important Cys residues. The enzyme’s two active sites occur at the interface between the R1 and R2 subunits. 
(b) A ribbon diagram of R22 viewed perpendicularly to its twofold axis with the subunits drawn in blue and yellow. The Fe(III) ions are represented by orange spheres, and the radical-harboring Tyr 122 side chains are shown in space-filling representation with their C
and O atoms green and red. 
(c) The binuclear Fe(III) complex of R2. Each Fe(III) ion is octahedrally coordinated by a His N atom and five O atoms, including those of the O2 ion and the Glu carboxyl group (a) (b) that bridges the two Fe(III) ions.

The most difficult step during RNR-catalyzed ribonucleotide reduction is the initial activation of a chemically unreactive C–H bond, and the only pathway known to date involves a cysteinyl radical capable of abstracting the 3’ H-atom from the ribose moiety 38

Ribonucleotide reduction takes place in four basic steps
the first of which involves activation of the substrate through abstraction of a hydrogen atom at the 3' position of the ribose. Subsequently, the 2' OH-group leaves as water, the substrate is reduced with two electrons and the initially abstracted 3' hydrogen is returned to the substrate to form the complete product. 26

The reaction catalyzed by RNR is strictly conserved in all living organisms.  A somewhat unusual feature of the RNR enzyme is that it catalyzes a reaction that proceeds via a free radical mechanism of action. The substrates for RNR are ADP, GDP, CDP and UDP. dTDP (deoxythymidine diphosphate) is synthesized by another enzyme (thymidylate kinase) from dTMP (deoxythymidine monophosphate). 

We have been able to make significant advances towards solving the long-standing problem of nucleotide abiogenesis, but our results highlight a number of issues that demand further investigation. First, what chemistry could have furnished the necessary chemical precursors, most importantly enantiomerically enriched glyceraldehyde? 2

The origins of homochirality and nucleotides seem to be inherently linked at the level of glyceraldehyde. As a racemizable molecule with one stereogenic center, glyceraldehyde
would appear to be the ideal molecule through which to provide at least enantiomeric enrichment or, ideally, dynamic kinetic resolution. Elucidating the origin of chiral glyceraldehyde and demonstrating the sequential sequestration of glycolaldehyde and glyceraldehyde or the separation/sanitization of ribonucleotide precursors may lead to the discovery of the chemical transformations that led to homochiral nucleotides.

Nucleotides must be oligomerized to generate RNA, and assuming that RNA must be 5′-3′-linked, a significant issue of regioselectivity must be overcome. Though, as a result of ringstrain, and are activated relative to NMP’s, they are not specifically activated to 5′-3′-oligomerization.

When DNA is randomly polymerized in prebiotic type experiments, you end up getting a mix dominated by 2′-5′ over 3′-5′ links, whereas for DNA to be readable it has to be uniformly 3′-5′. Without the homogeneity of homochirality, identical kinds of links, etc., even if life could have miraculously formed without all this, it would quickly self-destruct for the next generation. 34

Mechanisms of radical generation and transport
In spite of the related overall and active-site structures, the three RNR classes use different solutions to store and transfer the catalytically essential radicals.

Class I 
Class I RNR is an oxygen-dependent enzyme, which contains a dimetal-oxygen cluster (e.g. two u-oxo-bridged FeIIIFeIII clusters in class Ia) and, in most cases, include a nearby tyrosyl radical (∼5–6A˚ ). Class I RNRs possess a stable tyrosyl radical adjacent to a diiron cluster.  The radical is generated in the R2 subunit, where a tyrosyl radical results from hydrogen abstraction by an oxo-bridged diiron center, located 30– 40 A˚ from the active site. 48 The radical is subsequently transmitted through a long chain of hydrogen-bonded amino acids, from Tyr122 near the diiron complex to Cys439 at the active site in R1

The communication between the cysteine, Cys439, at the substrate site and the tyrosyl radical, Tyr122, in ribonucleotide reductase is studied by quantum chemical models 49  The α2 subunit houses the catalytic cysteine (C439) as well as two allosteric sites that control both substrate specificity and turnover rate. β2 stores a diiron-centered tyrosyl radical cofactor (Fe2–•Y122) that is essential for catalysis in α2. The binding of substrate and effector enhances inter-subunit interactions and triggers Fe2–•Y122 mediated C439 oxidation in α2 from a distance of 35 Å. Whereas the mechanism of catalysis by the enzyme is understood, the basis for S/E mediated conformational change and the attendant dynamic transport of the radical through the protein to the active site is yet to be unraveled. 50

At 35 Å separation, the vanishingly small overlap of the amino acid wave functions precludes ( make impossible )a single-step superexchange mechanism.  Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs).   NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 A across the ˚ interface of the two required subunits (b2 and a2) involving multiple Proton-coupled electron transfer (PCET) steps and the conserved tyrosine triad [Y356(b2)–Y731(a2)–Y730(a2)]. Catalysis by the class I RNRs proceeds by a radical mechanism requiring coupling of radical transport over 35 A involving PCET across ˚ the two subunits to substrate turnover. The long distance, reversibility, and rate-limiting conformational gating of radical transport have made study of this process challenging. The results suggest the importance of a well-coordinated proton exit channel involving Y356 and Y731 as key interfacial residues for radical transport across the a2:b2 interface. 51

Activation of Class I Ribonucleotide Reductases 
 In all classes, the post-translational activation of the enzyme consists of the specific generation of a protein radical. This radical serves for the abstraction of the hydrogen atom at the 3' position of the ribose moiety, which is a prerequisite for ribonucleotide reduction. The function of the iron center is in the formation of the tyrosyl radical cofactor. Class Ia ribonucleotide reductases (RNRs) are di-iron enzymes.  All class Ia RNRs utilize a diferric-tyrosyl radical (FeIII2-Y•) metallocofactor to catalyze the nucleotide reduction reaction. Subunit β houses the FeIII2-Y• cofactor that is assembled from FeII, O2, and a reducing equivalent. 58

A docking model of the E. coli α2β2 complex.2 α2 (pink and red) contains three nucleotide binding sites. 
β2 (light and dark blue) contains the diferric-Y• cofactor; residues 340 to 375 are not resolved in this structure. A peptide corresponding to the C-terminal 20 amino acids of β is bound to each α, a portion of which (residues 360-375) is resolved in the crystal structure (cyan). The “ATP cone” region of α, which contains the effector site that governs activity, is colored orange. This model separates Y122• in β2 from C439 in α2 by >35 Å. GDP (green), TTP (yellow) and the Fe2O core of the diferric cluster (orange) are shown in CPK space-filling models. Residues constituting the RT pathway (green) are shown in sticks. 58

The prototypical class Ia RNR from E. coli is composed of two subunits, α2 and β2, and is active as an α2β2 complex.α2 houses the catalytic site for substrate (S) reduction and two allosteric effector (E = ATP, dGTP, TTP, and dATP) binding sites that govern which S is reduced (specificity site) and the overall rate of reduction (activity site). β2 contains the essential diferric-Y• cofactor. This unusually stable Y•, located at position 122, has a half life of 4 days. Nucleotide reduction occurs by a complex mechanism involving protein- and substrate-derived radicals, some details of which are summarized in Figure below.

Mechanism of NDP reduction by RNR.
The S• shown on C439 of α2 in the first reaction step is reversibly generated by Y122• in β2 by the mechanism shown below:

(a) The proposed movement of protons (blue arrows) and electrons (red arrows) at each step on the pathway. Distances (Å) are from structures of α2 and β2. E350 and Y356 are disordered in all β2 structures, and their positions are unknown. There is no direct evidence that W48 and D237 participate in Radical Transfer and thus they are shown in gray. The distance between W48 and Y731 is modeled to be 25 Å. 
(b) The proposed relative reduction potentials of residues on the Radical Transfer pathway from experiments using the indicated UAAs site-specifically incorporated in place of each Y. NH2Y has been incorporated at position 356, 730, or 731. Note that the absolute reduction potentials of these residues, the structures, are not known, and the relative reduction potentials indicated are our best estimates given current knowledge.

The stable Y122• transiently oxidizes a cysteine (C439) in the catalytic site to a thiyl radical (S•), which reversibly abstracts a 3′-hydrogen atom (H•) from the NDP. The 3′-nucleotide radical rapidly loses water in the first irreversible step. The reducing equivalents are provided by two local cysteines (C225 and C462), and the resulting disulfide is re-reduced for subsequent turnovers, ultimately by thioredoxin (TR), thioredoxin reductase (TRR), and NADPH.

The diferric-tyrosyl radical cluster of ribonucleotide reductase
How each metalloprotein assembles the correct metal at the proper binding site presents challenges to the cell. Given the abundance of metalloproteins in the prokaryotic and eukaryotic proteomes, a main challenge for the cell is to load the Fe2+ onto the proper binding site and convert it into the active forms. 56

The existence of RNR is a pre-requisite for the existence of DNA, on which life, and biological Cells depend. So the assembly of metalloproteins and the correct metal at the proper binding site requires an explanation of what possible mechanism explains best its origin. It might be a challenge to random unguided self-assembly through prebiotic spontaneous chemical reactions by orderly aggregation after numerous trial and error attempts, in a sequentially correct manner without external direction. To a super-natural, super intelligent agency, it might be no challenge at all. 

Escherichia coli class-I RNR enzymes serve as a model of study of class-I RNR enzymes. These RNRs comprise two homodimeric subunits: R1 and R2. R1 is the center where the complex nucleotide-reduction process occurs, and the subunit serves as a paradigm for the secondary and tertiary structures adopted by the active sites of all classes of RNR. R2 contains the diiron(III)–tyrosyl-radical (Tyr) cofactor. The Tyr has unusual chemical stability ( half-life is 4 days ) and is essential to the nucleotide-reduction process. The class-I-RNR diiron metal cluster and amino-acid free radical provide a model for the mechanism of metal-cofactor assembly and the basis for the differential chemical reactivities of a wide range of proteins that require similar diiron clusters for catalysis.

The radical transfer from R2 to R1
The distance between the tyrosyl radical in R2 to the active cysteines in R1 is estimated to ∼35A˚. This distance is thus far beyond the reach of a pure electron tunnelling process. For this reason, it has been proposed that the radical is transferred by proton-coupled electron transfer (PCET), mediated by a chain of conserved hydrogen bonded amino acids that form transient radicals using an “electron relay” mechanism (Figure below)

The proposed radical transport pathway in E. coli RNR connecting the radical site Tyr-122 in R2 with the substrate binding site in R1. Coordinates obtained from PDB code 1MXR, chain A, and PDB code 4R1R, chain A . Y356 on the flexible tail of R2 is not resolved in the X-ray structure.

The tyrosyl radical site Y122 in R2 (using the E. coli numbering) and the thiyl radical site C349 in R1 are linked by a pathway of hydrogen bonds made by conserved amino acid side chains; Y730 andY731 in R1 and D84, H118, D237, and W48 in R2 [63,68]. The aromatic amino acid residues, including W48 and Y356 in R2 and Y371 and Y730 in R1, are conserved in all class I RNRs. When any of these tyrosine residues were substituted by phenylalanine, the activity of RNR is drastically decreased. The tyrosine residue Y356, located in the C-terminal tail of R2, is not resolved in the crystal structure, but it is one of the active components of the electron-transfer pathway and connects R2 to Y730 in R1. When this tyrosine residue was then replaced by mono- and polyfluorinated tyrosines (to systematically vary the radical-reduction potential and pKa on this pathway position) the catalytic activities and their pH dependency of the emerging protein variants, made it possible to determine the range of reduction potential required for a functional PCET. The results disclosed that Y356 was not mandatory for shuttling electrons. The R236 group plays a significant role in the radical transfer. It is hydrogen bonded to W48 in the R2 subunit provided by a structurally conserved water molecule and is conserved in the R2 proteins. Further studies have shown that mutation of this residue, resulted in that the active protein complex had a significant loss of catalytic activity.  57 

Studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for Radical Transfer 58

This is remarkable, as it examplifies how just one amino acid residue can determine if a fundamental enzyme reaction, in our case, radical transfer to the reaction site, is viable or not.

RNR co-factors and their synthesis

Class I RNR clusters
The class I ribonucleotide reductases (RNRs) require dinuclear metal clusters for activity 20

FeIIIFeIII-tyrosyl radical (Y•) cofactor (class Ia),
MnIIIMnIII-Y• cofactor (class Ib),
MnIVFeIII cofactor (class Ic).

2. Biochemistry, 8th ed. Styer, page  753
3. Biochemistry, 6th ed. Garrett, page 946
12. Life's greatest secrete, page 178
13. Encyclopedia of life ciences, page 5063
17. Enzyme-Catalyzed Electron and Radical Transfer, page 358
42. Biochemistry, 6th ed. Garrett, page 631
43. The Origin and Evolution of Ribonucleotide Reduction  Published: 27 February 2015

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Biosynthesis of RNR Class I (1a) diiron(III)–tyrosyl-radical (Tyr) cofactor
Nucleotide reduction by class Ia RNR requires a diferric-tyrosyl radical cofactor. The conserved Fe-S protein–diflavin reductase complex, Dre2–Tah18, plays a critical role in RNR cofactor biosynthesis. Cells with diminishing Dre2 have significantly reduced ability to make deoxynucleotides.   53 The β subunit houses a di-iron center that generates and maintains a tyrosyl radical (Y•), which is essential to initiate nucleotide reduction in the catalytic site of the α subunit via a long-range radical transfer pathway.  In response to DNA damage, an activated Mec1–Rad53–Dun1 checkpoint kinase cascade increases RNR levels by phosphorylation-dependent removal of Crt1, the transcriptional repressor of RNR2/3/4. 

The role of Dre2–Tah18 in RNR cofactor biosynthesis.
(A) The proposed pathways for biosynthesis and maintenance of the FeIII2-Y• cofactor of class Ia RNR. The biosynthetic pathway requires delivery of two FeII/β and a reducing equivalent to carry out the four-electron reduction of O2 to H2O; the other three electrons come from the two FeII and Tyr residue to form the FeIII2-Y•. The maintenance pathway may use the same source of reducing equivalents to convert the inactive FeIII2-Y cluster to FeII2-Y, which subsequently forms FeIII2-Y• in the presence of O2 via the biosynthesis pathway. 
(B) A model depicting the central role of the Dre2–Tah18 complex as a source of reducing equivalent for cluster assembly in RNR and cytosolic and nuclear Fe-S cluster-containing proteins. Assembly of FeIII2-Y• in β is facilitated by β′, which stabilizes β in a conformation that allows iron binding. Grx3/4 functions in intracellular iron trafficking and is required for most iron-requiring pathways, including the biosynthesis of Fe-S clusters in the mitochondria (ISC) and cytosol (CIA) and FeIII2-Y• assembly in RNR. Electrons from NADPH are transferred via FAD and FMN, the two flavin cofactors in Tah18, to the Fe-S cluster(s) in Dre2, which subsequently deliver the electrons to proteins in the CIA pathway and β in RNR. Dre2–Tah18 also may provide reducing equivalents to facilitate iron release from the [2Fe2S]-GSH2 cluster in the Grx3/Grx4 dimer.

An additional layer of RNR regulation, given that the level of Y• of the FeIII2-Y• cofactor is directly correlated with nucleotide reduction activity, involves the assembly and maintenance of this essential cluster. One very important set of modifications, often overlooked, is the assembly of metallocofactors essential for enzymatic activity. An estimated one-third of proteins require metals for their function.  recent studies from many groups have established the importance or essentiality of biosynthetic pathways for cluster insertion and have suggested the importance of repair or maintenance pathways for regenerating active cofactors from damaged metal clusters. A number of generalizations are emerging concerning nature’s design and implementation of metallocofactor biogenesis.

Nature, or the designers design?

The cofactors of many metalloproteins are  generated by defined biosynthetic pathways. ( How were theses pathways defined ? )
Metals are transferred in their reduced state to facilitate ligand exchange between protein factors.
Specific protein factors include a metal insertase or chaperone to deliver the metal, specific redox proteins such as flavodoxins or ferredoxins that control the oxidation state of the metal, and GTPases or ATPases involved in protein unfolding/refolding to allow metal entrance into deeply buried active sites. ( Metal insertases can be viewed as protein machines used to make parts used in other molecular machines, which are required to make the basic building blocks of life )
There is often biological redundancy in pathway factors (e.g., multiple ferredoxins)  ( Which promotes robustness and diminishes synthesis mistakes. Could nonguided random mechanisms explain this feat ? ) 
There is a hierarchy of metal delivery to proteins. ( How was this hierarchy established ? ) 
Compartmentalization (e.g., periplasm versus cytosol in prokaryotes) and relative affinities of protein coordination environments for various metals in relation to the intracellular concentrations of those metals contribute to prevention of mismetallation. ( How did this coordination emerge ? ) 
Metal clusters can become damaged by oxidants such as NO and O2•−, and specific pathways are implicated in their repair. ( How did these repair mechanisms emerge prebiotically ? ) 

Is the diiron(III) cluster of ribonucleotide reductase reduced and oxidized during turnover?
In the E. coli reductase, the nucleotide substrate binds on the second subunit (R1) and while, presumably, Tyr on R2 is oxidized and reduced every time a nucleotide is reduced to a deoxynucleotide, the diiron(III) cluster of R2 remains unchanged. However, if a mistake occurs, and the diiron(III)–tyrosyl-radical Tyr is reduced by an exogenous small molecule, a repair system regenerates the Tyr. Repair appears to involve reduction of the iron and, subsequently, the latter’s reaction with O2 and a reductant, which regenerates the active cofactor. Indeed, Tyr reduction/reoxidation could serve as one of many regulatory mechanisms for control of the deoxynucleotide-pool sizes – the maintenance of the relative ratios of deoxynucleotides being essential for DNA replication and repair.

Another paper reported the discovery of a 2Fe2S ferredoxin, YfaE in Escherichia coli and presented evidence that it plays a role in diferric-Y• cofactor maintenance and likely in diferric-Y• cofactor biosynthesis. 7 

Biosynthesis of RNR Class I (1b)

Class II is the coenzyme B12-dependent reductase
Class II enzymes are found in many microorganisms, both aerobes and anaerobes. They consist of a single polypeptide, either as a monomer or as a dimer, and use adenosylcobalamin (AdoCbl) to generate a cysteinyl radical. The availability of AdoCbl and/or SAM in the (pre)biotic soup must have played a key role in the determination of the first RNR. The complex pathway of AdoCbl generation has led to the suggestion that SAM is a ‘poor man’s’ AdoCbl and, hence, that the class III RNR is the likely progenitor. 

Organisms synthesize the complex organometallic framework of cobalamin ( Cbl )  using nearly 30 enzymatic steps in one of nature’s largest characterized biosynthetic pathways 6 Alternatively, organisms lacking the Cbl biosynthetic machinery can undergo the onerous task of salvaging Cbl from the environment using specific transport and chaperone systems. 

This is amazing. How could organisms evolve from scratch, salvage pathways?

Similarly, for organisms that uptake different forms of Cbl in their diet, extensive pathways for trafficking and processing these forms to a usable, biologically relevant state exist to ensure delivery of the correct cofactor to target enzymes. Unlike Cbl, the biosynthesis of AdoMet requires only a single step.

This is remarkable and raises the question. Why would an enzyme evolve the most complex co-factor, with the requirement of over nearly 30 enzymatic steps in one of nature’s largest characterized biosynthetic pathways for one Class ( RNR Class II ), and a co-factor requiring just one biosynthesis step for the other Class ( RNR Class III ), if both perform the same reaction?

Vitamin B12, also called cobalamin, is a water-soluble vitamin that is involved in the metabolism of every cell of the human body: it is a cofactor in DNA synthesis and in both fatty acid and amino acid metabolism. It is particularly important in the normal functioning of the nervous system via its role in the synthesis of myelin, and in the maturation of developing red blood cells in the bone marrow 

The class II RNR has been found mainly in bacteria and archaea. Both monomeric and dimeric forms have been described. The catalytic thiyl radical is formed by homolytic cleavage of the C-Co bond of the cofactor, 5′-adenosylcobalamin. This class of RNR has been reported to be common among cyanobacteria. 4

Vitamin B12 is one of eight B vitamins; it is the largest and most structurally complicated vitamin. It consists of a class of chemically related compounds (vitamers), all of which show physiological activity. It contains the biochemically rare element cobalt (chemical symbol Co) positioned in the center of a corrin ring. The only organisms to produce vitamin B12 are certain bacteria and archaea. Some of these bacteria are found in the soil around the grasses that ruminants eat; they are taken into the animal, proliferate, form part of their gut flora, and continue to produce vitamin B12. 1

Structure of 5′-deoxyadenosylcobalamin (coenzyme B12)

Structure of coenzyme B12. 
Coenzyme B12 is a class of molecules that vary, depending on the component designated X in the left-hand structure. 59-Deoxyadenosylcobalamin is the form of the coenzyme in methylmalonyl mutase. Substitution of methyl and cyano groups for X creates methylcobalamin and, cyanocobalamin, respectively.

The core of cobalamin consists of a corrin ring with a central cobalt atom (Figure above). The corrin ring, like a porphyrin, has four pyrrole units. Two of them are directly bonded to each other, whereas the others are joined by methine bridges, as in porphyrins. The corrin ring is more reduced than that of porphyrins and the substituents are different. A cobalt atom is bonded to the four pyrrole nitrogens. The fifth substituent linked to the cobalt atom is a derivative of dimethylbenzimidazole that contains ribose 3-phosphate and aminoisopropanol. One of the nitrogen atoms of dimethylbenzimidazole is linked to the cobalt atom. In coenzyme B12, the sixth substituent linked to the cobalt atom is a 5' deoxyadenosyl unit or a methyl group . This position can also be occupied by a cyano group. Cyanocobalamin is the form of the coenzyme administered to treat B 12 deficiency. In all of these compounds, the
cobalt is in the +3 oxidation state. 2

The biosynthesis of adenosylcobalamin (vitamin B12 ) 
Vitamin B12, or cobalamin, is one of the most structurally complex small molecules made in Nature. 3 The vitamin is a molecule of enormous complexity, containing a ring-contracted porphinoid with the cobalt ion ligated at the centre of the tetrapyrrole-derived macrocycle The cobalt ion is further held in place by a lower axial base (a dimethylbenzimidazole) and an upper cyano group. The cyano group is an artificial ligand, there as a result of the extraction procedure. In biological systems, two different upper axial ligands are found, either an adenosyl group or a methyl group, giving either AdoCbl or methylcobalamin. These are used in Nature to catalyse either rearrangement/reductase reactions (e.g. methylmalonyl CoA mutase and type II ribonucleotide reductases) or methyl transfer reaction such as those found in the metabolism of methanogenic bacteria or in cobalamin-dependent methionine synthesis. With the structure and function of biological forms of cobalamin now established, intense interest focused on how Nature was able to construct such a complex molecule. Vitamin B12 is exceptional in comparison to other vitamins and coenzymes for several reasons. Firstly, as alluded to earlier, there is its structural complexity, which is also reflected in its biosynthetic requirements such that somewhere around thirty genes are necessary for its complete de novo synthesis. Secondly, B12 is unique amongst the vitamins in that its synthesis is restricted to certain microorganisms. There is no genetic evidence that any eukaryote is able to make cobalamin. In contrast, in the prokaryotic world genome sequencing studies have revealed that the archae and certain eubacteria possess the genetic software that encode the cobalamin biosynthetic enzymes. 

Pathway comparisons. 
A comparison of the genes required for cobalamin biosynthesis between the “aerobic” and “anaerobic” pathways, which should more accurately be referred to as “late cobalt insertion” and “early cobalt insertion” pathways. The gene names and enzyme functions are given. Major genetic differences are highlighted in grey.

Branched pathway synthesis of modified tetrapyrroles. 
All modified tetrapyrroles, such as haem, chlorophyll, sirohaem, haem d1, coenzyme  F430 and vitamin B12 are derived from 5-aminolaevulinic acid (ALA). Two molecules of ALA are condensed to give the pyrrole porphobilinogen which is then transformed into the unsymmetrical type III isomer of uroporphyrinogen. The incorporation of ALA into both porphobilinogen and  uroporphyrinogen III is shown in colour. Uroporphyrinogen III can undergo decarboxylation and oxidation to give protoporphyrin IX, the  precursor for haem and chlorophyll, or can undergo methylation to produce precorrin-2, the progenitor for sirohaem, coenzyme F430, haem d1 and  vitamin B12.

The biochemical steps required for the transformation of uroporphyrinogen III into AdoCbl are one of the most mesmerizing and at times bewildering pathways operated in nature. It is outside the scope of this paper to cover aspects of control and regulation or to delve too deeply into how this intricate network of enzymes may have arisen. Similarly, we have not dealt with the biochemistry of the end product, the role that vitamin B12 plays in biological systems, the beguiling chemical transformations it is able to mediate. These aspects, harnessed with the many unanswered questions concerning its biosynthesis, can be addressed in future reviews on Nature’s most interesting and charismatic vitamin.

In class II RNRs the electron hole on AdoCbl-derived dAdo• is transferred to the 3' position of the substrate nucleotide via a cysteinyl radical intermediate. 5 Assuming an evolutionary process based on tinkering, i.e., modification of present components rather than inventions from scratch, our model of the protoRNR tries to deviate as little as possible from what is observed in modern biochemistry. 5

Amazing. An evolutionary process based on tinkering. Let that sink in for a moment.... How does such a proposal make sense? If the author wishes not to deviate much from the mechanisms observed in modern enzymes, he has to acknowledge for the origin of the most complex biosynthesis pathways to produce for example one of the most sophisticated cofactors known ( Vitamin B12 in RNR Class II enzymes ). That is not logical nor rational. An evident answer would be that intelligence with specific goals was involved, namely to create the most effective and advanced bioinformational molecule known to man: DNA.

2. Biochemistry 8th ed. Styer, page 655

Further details on Biosynthesis of Vitamin B12, see:
How Do Organisms Synthesize Amino Acids?

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7 RNR Class III [4Fe-4S] cluster and AdoMet on Sun Jun 21, 2015 10:07 am


RNR Class III [4Fe-4S] cluster and AdoMet biosynthesis
Class III anaerobic ribonucleotide reductase small component, named protein b, contains a (4Fe-4S) center. Its function is to mediate electron transfer from reduced flavodoxin to S-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein a, which then becomes active for the reduction of ribonucleotides. 36 The reductase, named protein a, in the form of a dimer a2, contains the active site where substrates and formate react and allosteric effectors bind.  It is activated during anaerobic incubation with a reducing system (NADPH+flavodoxin reductase+flavodoxin )  Protein bis an iron-sulfur protein, also sensitive to oxygen. Under strict anaerobic and reductive conditions it can assemble a (4Fe-4S) center. Protein beta, essential during anaerobic activation of protein abecause it catalyzes the oneelectron transfer from reduced flavodoxin to AdoMet required for the formation of the glycyl radical. As a matter of fact it has been shown that the reduced (4Fe-4S)1cluster is able to reduce AdoMet, and it is postulated that this reaction results in the homolytic cleavage of its S-C(5'-deoxyadenosyl) bond and formation of a 5'-deoxyadenosyl radical, responsible for H atom abstraction at the specific glycine residue. The combination of an iron-sulfur center and AdoMet for generating free radicals appears to be a general strategy in biological systems.

RNR uses a tyrosyl radical of protein R2, the first radical site is generated in protein R2 in a reaction involving molecular oxygen and a diiron site
class II, a cobalamin derived adenosyl radical, cobalamin binds directly to the catalytic subunit and 
class III, a glycyl radical. All are ultimately responsible for the generation of a thiyl radical on a structurally conserved cysteine residue e  at the active site.  Class I enzymes require Oxygen O2 for radical generation and employ a di-iron cluster on a separate subunit (R2) to produce a stable tyrosyl radical. Long-range electron transfer from the catalytic thiol to the tyrosyl radical of R2 generates the thiyl radical. Since oxygen was presumably absent from the atmosphere at the time of the transition from the RNA world to the DNA world, this enzyme class is thought to be an evolutionary latecomer. The other two classes of ribonucleotide reductase can generate the catalytic thiyl radical under strictly anaerobic conditions. 

Class II enzymes use the adenosylcobalamin form of the B12 cofactor (AdoCbl); cleavage of the weak carbon-cobalt bond of AdoCbl forms a 5′-deoxyadenosyl radical that in turn generates the thiyl radical (Figure below).

Formation of the thiyl radical in class II ribonucleotide reductases. 
Homolysis of the Co– 5’ CH2 bond, enhanced by addition of effector11, forms the adenosyl radical that abstracts the hydrogen from cysteine

These enzymes have only one type of subunit and are monomers or homodimers.

Class III enzymes use S-adenosylmethionine (AdoMet) to generate a radical on a glycyl residue. The glycyl radical is formed by reaction with a 5′-deoxyadenosyl radical derived from AdoMet in a step that requires a second subunit containing an iron–sulfur cluster and uses a reducing equivalent provided by flavodoxin.

Reduction occurs through the intermediacy of protein thiols that are oxidized concomitant with substrate reduction. Reducing equivalents are first transferred from NADPH to oxidized thioredoxin by thioredoxin reductase, and then on to the thiol groups of ribonucleotide reductase. 17 The role of AdoCbl c  in the reduction process is to initiate the formation of a protein thiyl radical d. The thiyl radical activates the substrate towards reduction by abstracting the 3'-hydrogen atom of the ribonucleotide

A tyrosyl radical is critical to the action of ribonucleotide reductase
The ribonucleotide reductase of E. coli consists of two subunits: R1 and R2. The R1 subunit contains the active site as well as two allosteric control sites. This subunit includes three conserved cysteine residues and a glutamate residue, all four of which participate in the reduction of ribose to deoxyribose (Figure below).

Ribonucleotide reductase. 
Ribonucleotide reductase reduces ribonucleotides to deoxyribonucleotides in its active site, which contains three key cysteine residues and one glutamate residue. Each R2 subunit contains a tyrosyl radical that accepts an electron from one of the cysteine residues in the active site to initiate the reduction reaction. Two R1 subunits come together to form a dimer as do two R2 subunits.

The R2 subunit’s role in catalysis is to generate a free radical in each of its two chains. Each R2 chain contains a tyrosyl radical with an unpaired electron delocalized onto its aromatic ring , generated by a nearby iron center consisting of two ferric (Fe 3 + ) ions bridged by an oxide (O 2– ) ion

The best-characterized example of type of iron metalloenzyme is ribonucleotide reductase, in which a binuclear iron(II) center reacts with oxygen to produce a binuclear iron(III) center and a tyrosyl radical, the latter being an absolute requirement for catalytic activity. Iron, in the form of an iron-sulfur cluster, is also centrally involved in the generation of the glycyl radical in ribonucleotide reductase from anaerobic E. coli and the glycyl radical of pyruvate formate-lyase; again, both of these radicals are catalytically essential. 13

Regulation of the right amount of DNA production
Nucleotide biosynthesis is regulated by feedback inhibition in a manner similar to the regulation of amino acid biosynthesis. These regulatory pathways ensure that the various nucleotides are produced in the required quantities. Allosteric regulation of RNR's serves to maintain an appropriate supply of each of the four deoxynucleotides (dNTPs) needed for DNA replication and repair. This regulation prevents dNTP imbalances that can be genotoxic and lead to high mutation rates. 64

How could the Cell survive without this regulation? This raises the question: Could the regulation mechanism have emerged slowly, gradually, and step by step, if, not fully setup, mutation rate would kill the Cell? 

A sophisticated allosteric regulation manifested by the binding of deoxyribonucleoside triphosphates and ATP allows the enzyme to coordinate a balanced production of precursors for DNA replication and repair. 60

Ribonucleotide reductase activity must be modulated in two ways in order to maintain an appropriate balance of the four deoxynucleotides essential to DNA synthesis, namely, dATP, dGTP, dCTP, and dTTP. First, the overall activity of the enzyme must be turned on and off in response to the need for dNTPs. Second, the relative amounts of each NDP substrate transformed into dNDP must be controlled so that the right balance of dATP;dGTP;dCTP;dTTP is produced. The two different allosteric sites on ribonucleotide reductase, discrete from the substrate-binding catalytic site, are designed to serve these purposes. These two allosteric sites are designated the overall activity site and the substrate specificity site. Only ATP and dATP are able to bind at the overall activity site. ATP is an allosteric activator and dATP is an allosteric inhibitor, and they compete for the same site. If ATP is bound, the enzyme is active, whereas if its deoxy counterpart, dATP, occupies this site, the enzyme is inactive. Although ATP is more abundant than dATP, dATP binds to ribonucleotide reductase with 100-fold greater affinity. When dATP binds, R1 dimers aggregate to form inactive hexamers; ATP binding reverses this oligomerization. The second allosteric site, the substrate specificity site, can bind either ATP, dTTP, dGTP, or dATP, and the substrate specificity of the enzyme is determined by which of these nucleotides occupies this site. If ATP is in the substrate specificity site, ribonucleotide reductase preferentially binds pyrimidine nucleotides (UDP or CDP) at its active site and reduces them to dUDP and dCDP. With dTTP in the specificity-determining site, GDP is the preferred substrate. When dGTP binds to the specificity site, ADP becomes the favored substrate for reduction. The rationale for these varying affinities is as follows (Figure below): 

Regulation of deoxynucleotide biosynthesis: 
the rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotide reductase.

High [ATP] is consistent with cell growth and division and, consequently, the need for DNA synthesis. Thus, ATP binds in the overall activity site of ribonucleotide reductase, turning it on and promoting the production of dNTPs for DNA synthesis. Under these conditions, ATP is also likely to occupy the substrate specificity site, so UDP and CDP are bound at the catalytic site and reduced to dUDP and dCDP. Both of these pyrimidine deoxynucleoside diphosphates are precursors to dTTP. Thus, elevation of dUDP and dCDP levels leads to an increase in [dTTP]. High dTTP levels increase the likelihood that it will occupy the substrate specificity site, in which case GDP becomes the preferred substrate and dGTP levels rise. Upon dGTP association with the substrate specificity site, ADP is the favored substrate, leading to ADP reduction and the eventual accumulation of dATP. Binding of dATP to the overall activity site then shuts the enzyme down. In summary, the relative affinities of the three classes of nucleotide-binding sites in ribonucleotide reductase for the various substrates, activators, and inhibitors are such that the formation of dNDPs proceeds in an orderly and balanced fashion. As these dNDPs are formed in amounts consistent with cellular needs, their phosphorylation by nucleoside diphosphate kinases produces dNTPs, the actual substrates of DNA synthesis.

RNR plays a critical role in regulating the total rate of DNA synthesis so that DNA to cell mass is maintained at a constant ratio during cell division and DNA repair. One of the most important aspects of the Deoxynucleotide (dNTP) supply required for DNA synthesis and repair is the tight regulation of RNR's at different levels, including the allosteric regulation of enzyme activity, transcriptional regulation, and cell cycle-specific proteolysis in mammalian cells. 

RNR activity is controlled at two different levels: substrate specificity, in which the binding of different nucleotides results in the reduction of each specific NTP at the active site, and enzymatic activity, in which the binding of ATP, or dATP respectively activates or inhibits enzymatic activity.

Schematic overview of the two modes of allosteric regulation in ribonucleotide reductases.
The overall activity is governed by the binding of dATP (inhibition) or ATP (stimulation) to the activity site, located in a small N-terminal ATP cone domain of the α2 subunit of RNRs from class Ia and III. With very few exceptions, class II RNRs lack ATP cones and are not activity regulated. The substrate specificity is regulated by the binding of dNTPs to the specificity site at the dimer interface: ATP and dATP upregulate the reduction of CDP and UDP, dTTP upregulates GDP reduction and dGTP increases the rate of ADP reduction. Loop 2 is a flexible loop involved in transmission of the specificity signal. 45

Regulation of ribonucleotide reductase.
(A) Each subunit in the R1 dimer contains two allosteric sites in addition to the active site. One site regulates the overall activity and the other site regulates substrate specificity.
(B) The patterns of regulation with regard to different nucleoside diphosphates demonstrated by ribonucleotide reductase. 

RNR is considered to be the master regulator, as verified by numerous studies of mammalian and yeast cell lines where its allosteric sites have been mutated. Many of these cell lines have severely skewed dNTP levels and dramatically increased mutation rates 44 The synthesis and regulation of the four dNTPs is a sophisticated task and, despite RNR having attained a “scientific age” of over 50 years, research on this enzyme continues to uncover new and fascinating aspects. In particular, two recent studies have contributed intriguing advances in our understanding of the intricacies and complexities of its allosteric regulation.  Remarkably, the overall pattern of allosteric regulation is the same across all three classes, despite their different radical chemistries and quaternary associations. We do not yet understand how the pattern has been conserved over large evolutionary distances.

When a cell copies its DNA, it uses four different building blocks deoxyribonucleotides (dNTPs). These consist of one of the four ‘bases’ (A, T, C and G), which pair up to link the two strands of DNA in the double helix, bound to a sugar and a phosphate group. If the cell contains too little or too much of one of these building blocks, an incorrect base may be inserted into the DNA. This results in a mutation, which in bacteria can cause death, and in animals may lead to cancer.

The enzyme that fabricates and carefully controls the amount of each dNTP building block inside a cell is called ribonucleotide reductase. Once there are enough building blocks in a cell the enzyme is turned off. A part of the enzyme called the ATP-cone acts as an on/off switch to control this activity. 24 When the amount of dNTP building blocks reaches a certain limit, the ATP-cone turns off the enzyme. When turned off, the enzyme’s small components are glued together in pairs. This prevents them from working.  

Allosteric regulation of an enzyme is defined as regulation of activity by binding of an effector molecule to a different location of the enzyme than the active site. The effector influences the distribution of tertiary or quaternary conformations of an enzyme, alone or in combination, modulating its activity.  Allostery is an intrinsic property of many, if not all, dynamic proteins .

Many RNRs possess an overall activity regulation site (a-site) positioned in an N-terminal domain of ~85–100 amino acid residues called the ATP-cone

Structure of the ATP-cone domain 
The ribbon diagram of the NrdA  ATP-cone domain. The conserved residues that participate in ATP contacts and are indicated by asterisks are shown in a ball-and-stick representation along with the ATP molecule. The hydrogen bonds formed by these residues with ATP are indicated by dashed lines. 25

Acting as a regulatory master switch, the a-site senses intracellular nucleotide concentrations by competitive binding of ATP and dATP. In the presence of ATP the enzyme is active, and when concentrations of dNTPs rise, binding of dATP switches the enzyme off. This mechanism ensures sufficient but not excessive amounts of nucleotides that may also cause increased mutation rates. The ATP-cone is an example of allosteric regulation controlled by a domain that acts relatively independent of the catalytic core of proteins.

NrdA and NrdB interact to form the active complex, in which the two proteins need to be precisely positioned such that the radical can be transferred from NrdB, where it is generated and stored, to NrdA, where it starts the substrate reduction.

Ribonucleotide reductase (RNRs) enzymes are largely responsible for the regulation of the concentrations and relative ratios of the Deoxynucleotides, which govern the fidelity of DNA replication and repair. RNRs are regulated at many levels 20 

Nordlund and Reichard published in 2006 an article about Ribonucleotide Reductases, and in regards of DNA production regulation, they wrote:
An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, together with the allosteric effects, enzyme activity and provides the appropriate amount of deoxynucleotides for DNA replication and repair. A common feature of all reductases is their ability to provide an appropriate balance of the four DNA building blocks. A unique allosteric regulation of their substrate specificity makes this possible. All RNRs share a common basic catalytic mechanism involving the activation of the ribonucleotide by abstraction of the 3'- hydrogen atom of the ribose by a transient thiyl radical of the enzyme. RNR activity is cell cycle related and highest early in the exponential phase in bacteria and during S phase in eukaryotes, when the requirement for dNTPs is largest. 22

RNR produces the correct amount of each of the four dNTPs because Nature invented diverse intricate mechanisms to regulate gene transcription, protein degradation, messenger RNA stability, and specific inhibitors, as well as a unique allosteric control of enzyme activity.

Nature invented? Wiki describes inventing as “come upon, meet with, find, discover”. The verb englobes: To design a new process or mechanism. To create something fictional for a particular purpose. To come upon; to find; to discover. 23 Without any doubt, invent is something that only a mind, intelligence, a person can do. Nature "per se" is nothing of this. The attempt to smuggle teleological terms into biology extends through all biology textbooks and demonstrates how it is difficult for the authors to leave teleology out of the door. But, if we contemplate the complexity of the described processes, how could we describe them without invoking these terms? It is evident that these processes point to the requirement of inventive intelligence for implementation, which points to a creative agency.

When it comes to how RNR's emerged, the authors continue: It is generally believed that RNA preceded DNA during the evolution of life and therefore that ribonucleotides existed before deoxyribonucleotides. With the appearance of ribonucleotide reduction, DNA could replace RNA as the repository of genetic information. Ribonucleotide reduction involves radical chemistry that is not likely to be catalyzed by RNA but should require the shielded environment of a protein. According to this line of thought, an RNR was a prerequisite for the appearance of DNA, and proteins preceded DNA during evolution.

The chicken - egg catch22 situation is evident. RNR's are required to make DNA, but DNA is required to make these enzymes. What came first? The only rational explanation is that they were created all at once by a creator. But the problem and conundrum goes further. The paper continues:

One might expect that an enzyme performing such an essential function would have been conserved. Instead, there are three separate classes, with widely diverging amino acid sequences and different mechanisms for the generation of the free radical required for catalysis. The three classes show, however, related three-dimensional structures and striking similarities in their complicated allosteric regulation as well as in the catalytic
mechanism.We have therefore suggested that the three classes arose by divergent evolution from a common ancestor that existed before the transition of the RNA to DNA world.

Why would a common ancestor produce the same molecular mechanism in three different routes?
Importance of the Maintenance Pathway in the Regulation of the Activity of Escherichia coli Ribonucleotide Reductase 21

The Inability of Oxidized Ribonucleotide Reductase to Bind Substrate Serves an Essential Protective Function. 
Comparison of the X-ray structures of reduced R1 (in which the redox-active Cys 225 and Cys 462 residues are in their SH forms) and oxidized R1 (in which Cys 225 and Cys 462 are disulfide-linked) reveals that Cys 462 in reduced R1 has rotated away from its position in oxidized R1 to become buried in a hydrophobic pocket, whereas Cys 225 moves into the region formerly occupied by Cys 462. The distance between the formerly disulfide-linked S atoms thereby increases from 2.0 Å to 5.7 Å. These movements are accompanied by small shifts of the surrounding polypeptide chain. R1 Cys 225 in oxidized ribonucleotide reductase prevents the binding of substrate through steric interference of its S atom with the substrate NDP’s O2' atom.

The inability of oxidized ribonucleotide reductase to bind substrate has functional significance. 
In the absence of substrate, the enzyme’s free radical is stored in the interior of the R2 subunit, close to its dinuclear iron center. When substrate is bound, the radical is presumably transferred to it via a series
of protein side chains in both R2 and R1. If the substrate is unable to properly react after accepting this free radical, as would be the case if the enzyme were in its oxidized state, the free radical could potentially destroy both the substrate and the enzyme. Thus, an important role of the enzyme is to control the release of the radical’s powerful oxidizing capability. It does so in part by preventing the binding of substrate while the enzyme is in its oxidized form.

Question: Would the enzyme not be destroyed in a trial and error process, in order to find the right configuration? 

Ribonucleotide Reductase Is Regulated by a Complex Feedback Network
The synthesis of the four dNTPs in the amounts required for DNA synthesis is accomplished through feedback control. Maintaining the proper intracellular ratios of dNTPs is essential for normal growth. Indeed, a deficiency of any dNTP is lethal, whereas an excess is mutagenic because the probability that a given dNTP will be erroneously incorporated into a growing DNA strand increases with its concentration relative to those of the other dNTPs. The catalytic activity of mouse ribonucleotide reductase varies with its state of oligomerization, which in turn is governed by the binding of nucleotide effectors to three independent allosteric sites on R1

The cofactors of many metalloproteins are likely generated by defined biosynthetic pathways. Metals are transferred in their reduced state to facilitate ligand exchange between protein factors. Specific protein factors include a metal insertase or chaperone to deliver the metal, specific redox proteins such as flavodoxins or ferredoxins that control the oxidation state of the metal, and GTPases or ATPases involved in protein unfolding/refolding to allow metal entrance into deeply buried active sites. There is  a hierarchy of metal delivery to proteins. Compartmentalization (e.g., periplasm versus cytosol in prokaryotes) and relative affinities of protein coordination environments for various metals in relation to the intracellular concentrations of those metals likely contribute to prevention of mismetallation. Many proteins are never isolated from their native source but instead from heterologous expression systems, often leading to insertion of incorrect metals. Because the “gold standard” of activity is unknown, low activity associated with incorrect clusters may go unrecognized. Metal clusters can become damaged by oxidants such as NO and O2•−, and specific pathways are implicated in their repair. Finally, during changes of oxidation state, ligands to the metal (e.g., His, Asp, Glu, and waters) can reorganize readily; structural rearrangements of carboxylate ligands (“carboxylate shifts”) are often critical to the cluster assembly process, and protons are often required for metal oxidation.

The diferric-tyrosyl radical cluster of ribonucleotide reductase
How each metalloprotein assembles the correct metal at the proper binding site presents challenges to the cell. 46

The existence of RNR is a pre-requisite for the existence of DNA, on which life, and biological Cells depend. So the assembly of metalloproteins and the correct metal at the proper binding site requires an explanation of what possible mechanism explains best its origin. It might be a challenge to random unguided self-assembly through prebiotic spontaneous chemical reactions by orderly aggregation after numerous trial and error attempts, in a sequentially correct manner without external direction. To a super-natural, super intelligent agency, it might be no challenge at all. 

Evolution of Ribonucleotide Reductases
The science paper:  Ribonucleotide reductases: the link between an RNA and a DNA world? 59 make a remarkable claim:
RNRs evolved and provide an essential link between the RNA and DNA world. These enzymes are essential for both DNA replication and repair.

Question: How could evolution be a driving force prior to the emergence of DNA, upon which evolution depends?

Assuming that a ribozyme will not be capable of catalyzing such complex radical chemistry, then proteins must have preceded DNA in the transition from an RNA to a DNA world.

This is a paradox. Proteins are encoded in DNA, and could not precede it. 

The availability of AdoCbl and/or SAM in the (pre)biotic soup must have played a key role in the determination of the first RNR. The complex pathway of AdoCbl generation has led to the suggestion that SAM is a ‘poor man’s’ AdoCbl and, hence, that the class III RNR is the likely progenitor. 

From a chemical point of view the relatively simple AdoMet can be viewed as a forerunner to the complex adenosyl cobalamin. I therefore propose that the "ur"- reductase operated with an AdoMet-based glycyl radical mechanism, similar to present-day class III enzymes. Such a glycyl radical mechanism is used by pyruvate formate lyase, a key enzyme of anaerobic metabolism. One can speculate that this was a basic mechanism, first developed for anaerobic energy metabolism during evolution. The "ur"-reductase might then have exploited it for its own purpose.

The appearance of oxygen on Earth required new radical generating mechanisms involving major restructuring of the protein. Two oxygen-tolerating mechanisms evolved, one based on adenosyl cobalamin (class II enzymes), the other on an Fe(III)- tyrosyl radical center (class I enzymes). Certain structural and mechanistic features suggest a relation between the three classes of reductases, but the strongest argument comes from their almost identical allosteric behavior.In conclusion, I propose that the three classes of ribonucleotide reductases have evolved from a common ancestor, in spite of large structural differences.  63

How could the author propose anything different that would make more sense, namely design by a common designer, if methodological naturalism restrains the range of explanations to natural scenarios and occurrences without intelligent intervention? RNR's are a nightmare for any proponent of evolution - not even mentioning, that the authors overlook the fact that RNR's had to emerge prior when life began, and so, before biological evolution. 

A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited
Nature’s most beautiful cofactor, cobalamin (Cbl), is a biosynthetically expensive, elaborate, and light-sensitive organometallic Co-containing compound  62 AdoCbl-dependent enzymes are instead traditionally used to catalyze radical-based reactions; Similar chemistry can be performed using a remarkably different cofactor, Sadenosylmethionine (AdoMet, or SAM). AdoMet is a simple molecule, built in a single step that results in the coupling of methionine with the adenosyl moiety of adenosine triphosphate (ATP) 

According to an article published in Science Magazine in 1993:
Probably the ability to reduce ribonucleotides had to exist at a time when both RNA and protein existed and before the appearance of DNA.  In recent years RNA has indeed shown unexpected catalytic abilities; however, assuming a requirement for radical chemistry, the risk of self-destruction of an RNA reductase appears considerable. RNA lacks the versatility of the protein structure that can be used by nature to contain radicals in hydrophobic pockets. 29

How and when ribonucleotide reduction evolved is a question that is intimately associated with the transition from the RNA world to the modern RNA + protein + DNA world, since it is the only known de novo mechanism for dNTP synthesis. The maintenance of life on Earth depends on the ability to reproduce. Reproduction requires an accurate and stable storage system for the genetic information in all organisms, including viruses. It has been recently suggested that the RNA molecule, with autoreplicative capacity, is the primary primitive molecule for the genetic information storage. Despite the wide acceptance of this idea, there are arguments against the concept of an RNA world that cannot be underestimated.

While it is entirely possible that a different pathway was later replaced with the modern mechanism, here we explore the evolutionary and biochemical limits for an origin of the mechanism in the RNA + protein world and suggest a model for a prototypical ribonucleotide reductase (protoRNR). From the protoRNR evolved the ancestor to modern RNRs, the urRNR, which diversified into the modern three classes. Since the initial radical generation differs between the three modern classes, it is difficult to establish how it was generated in the urRNR. Here we suggest a model that is similar to the B12-dependent mechanism in modern class II RNRs. 43

It is remarkable that the authors suggest an evolutionary mechanism prior to DNA replication. Biological evolution depends on DNA replication.  

Iron-Sulfur Clusters in Chemistry and Biology, page 258:
The conversion of ribonucleotides into deoxyribonucleotides is catalyzed by three evolutionarily unrelated ribonucleotide reductases (RNR). Each of the three enzymes requires a system to generate a free-radical species at its active site. Class III RNRs, which are considered the most ancient RNRs, use a radical SAM enzyme, and are therefore Fe-S dependent.

The author of this book makes the opposite claim, namely that the three RNR classes are unrelated. That demonstrates, there is no consensus about the origin of the divergences. 

Reaction Mechanism of the ProtoRNR
The appearance of powerful metal-catalyzed redox chemistry was a crucial step for the origin of ribonucleotide reduction. Arguably the most difficult step during RNR-catalyzed ribonucleotide reduction is the initial activation of a chemically unreactive C–H bond, and the only pathway known to date involves a cysteinyl radical capable of abstracting the 3’ H-atom from the ribose moiety.   The most common intermediate under anaerobic conditions is the 5' deoxyadenosyl 5' radical (dAdo•), i.e., a radical positioned on the dehydroxylated 5' carbon of the ribose ring of an adenosine. This species is generated either via homolytic cleavage of adenosylcobalamin (AdoCbl) or from S-adenosylmethionine (AdoMet) by single electron injection from an iron-sulfur cluster. 

Question: Had there not to be 1. the recognition of requirement of ribonucleotide reduction for the specific purpose to create a molecule, namely deoxyribonucleotides, able to polymerize into long stable information storing molecules?  Why would a prebiotic earth have the need to form such molecules without an apparent goal or need? And secondly, the know-how to perform the reaction to reach the goal, and its implementation? and how to synthesize the molecules and bring the individual subparts together and join them in the right assembly sequence? Had there not to be know-how of radical chemistry, and how to abstract the 3'H atom from the ribose moiety in the first of the four reactions required to conclude the task and reach the goal?  

It should also be noted that while the biosynthesis of both iron-sulfur clusters and metalloporphyrins (e.g., AdoCbl) requires complex machineries in modern organisms, iron-sulfur clusters can form spontaneously in vitro from relatively simple starting materials and abiotic porphyrin synthesis has been suggested. Moreover, both cobalt and iron are considered to have been bioavailable to a much greater extent prior to the great oxygenation event than they are today.

On the basis of structure and suggested sequence homology all three classes appear to share a common origin.

That claim stand in direct opposition to following:

Today, three different RNR classes have been described, with little apparent similarity between them in terms of primary protein sequence (approximately 10–20% similarity). Thus, it could be assumed that each RNR class appeared independently from each other over time. 37

All require complex protein radical chemistry to reduce ribonucleotides to deoxyribonucleotides, though the radical generation mechanism differs between the classes. The highly complex nature of the reduction suggests that catalytic proteins had to arise before the transition to DNA could occur. Catalytic RNA (ribozymes), almost certainly could not have carried out such free radical chemistry ^ even if a radical could have been generated it is unlikely it could have been controlled. A salient example of this is the use of radicals in probing of nucleic acid structure. Fe(II)^EDTA generated radicals cleave RNA and DNA non-speci¢cally, regardless of whether single-stranded or double-stranded, and irrespective of local structure. While class II and III reductases use nucleotide cofactors, this does not mean that the reactions would have occurred in the RNA world, though these cofactors may date from the RNA world. It is the cofactors themselves, not the diversity of reactions they are involved in, that date back to the RNA world ^ they have been recruited into many new metabolic pathways during evolution. Given known RNA chemistry, ribonucleotide reduction appears impossible until the development of protein catalysts.

2. Biochemistry, 8th ed. Styer, page  753
3. Biochemistry, 6th ed. Garrett, page 946
12. Life's greatest secrete, page 178
13. Encyclopedia of life ciences, page 5063
17. Enzyme-Catalyzed Electron and Radical Transfer, page 358
42. Biochemistry, 6th ed. Garrett, page 631
43. The Origin and Evolution of Ribonucleotide Reduction  Published: 27 February 2015
64. Biochemistry 8th ed. Styer, page 759

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8 How Are Thymine Nucleotides Synthesized? on Sun Jun 21, 2015 9:11 pm


How Are Thymine Nucleotides Synthesized?

RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a –CH3  ( methyl group ) Spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day, and one of the most common methods of damage. ( see below ) Had DNA not switched from uracil to thymine, the deamination damage to cytosine would be essentially impossible to detect. But since thymine is used by DNA, uracil can be correctly recognized as damaged and repaired back to cytosine with thymine as template.

Deamidation is by removing the amino groups of the molecule is the hydrolysis reaction of cytosine into uracil, releasing ammonia in the process. In DNA, this spontaneous deamination is corrected for by the removal of uracil ( which is the deterioration product of cytosine deamination and not part of DNA) by uracil-DNA glycosylase 3  generating an abasic (AP) site b.

Depurination and deamination. 
These reactions are two of the most frequent spontaneous chemical reactions that create serious DNA damage in cells. Depurination can release guanine (shown here), as well as adenine, from DNA. The major type of deamination reaction converts cytosine to an altered DNA base, uracil (shown here), but deamination occurs on other bases as well. These reactions normally take place in double-helical DNA; for convenience, only one strand is shown. 5

Nucleotide deamination: a transition mutation occurs: cytosine is converted to uracil (which base pairs with adenine). 2

Why do cells go to the trouble of methylating uracil to thymine before it can be used in DNA?

1, ribonucleoside reductase; 
2, nucleoside diphosphate kinase; 
3, dCMP kinase; 
4, dCMP deaminase; 
5, dUTPase; 
6, thymidylate synthase; 
7, thymidylate kinase. CH2 = THF is 5,10-methylenetetrahydrofolate, and DHF is dihydrofolate.

The thymine-uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson-Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to appear in DNA: thymine replacement and cytosine deamination. 20 Most DNA polymerases readily incorporate dUMP as well as dTMP depending solely on the availability of the d(U/T)TP building block nucleotides. Cytosine deamination results in mutagenic U:G mismatches that must be excised. The repair system, however, also excises U from U:A “normal” pairs. It is therefore crucial to limit thymine-replacing uracils.

De novo biosynthesis of thymine is an intricate and energetically expensive process that requires dUMP as the starting material and a complex array of two enzymes and cofactors.  It is therefore straightforward to ask: is there any specific reason that justifies this costly and seemingly equivalent replacement of uracil by thymine in DNA? It is generally accepted that negative discrimination against uracil in DNA is caused by the chemical instability of cytosine. Deamination of cytosine, a rather frequent process that readily occurs under physiological circumstances, gives rise to uracil .   Unless corrected, this mutagenic transition will result in a C:G into U(T): A base-pair change, that is, a stable point mutation. To deal with this problem, a highly efficient repair process (uracil-excision repair)  starts with uracil–DNA glycosylase (UDG). The importance of this repair process is well-reflected in two observations. One, cytosine deamination is one of the most frequent spontaneous mutations in DNA.Two, UDG activity resides in at least four families of enzymes: redundancy may be required for specific circumstances. 21 

During nucleotide synthesis,  ribonucleotides that form RNA, are transformed into deoxyribonucleotides that form DNA. Before being incorporated into the chromosomes, another essential modification takes place. Uracil bases in RNA are transformed into thymine bases in DNA. There is a life essential requirement, why. Cytosine, the second of the two pyrimidine bases used in DNA, do deaminate over time into uracil bases. If uracil would remain, and not be replaced by thymine in DNA,  it would mix with the cytosine which deaminated spontaneously into uracil -  occurring on average, 100 times per day in the cell. Deamination of cytosine into deoxyuridine (a common spontaneous chemical reaction) can lead to incorporation of numerous mutations in the chromosome during replication with disastrous outcomes. If there wasn't a mechanism to remove the deaminated nucleotides (dUMP's), then, gradually over time, all of the Cytosine-Adenine base pairings would become a Uracil-Adenine base pairing.

If uracil would be transformed from RNA to DNA, transforming it into deoxy uracil, and used in DNA like it is in RNA, and not replaced with thymine, then it would keep being recognized as deaminated cytidine by the repair machinery, and removed as well,  and the DNA would be basically coated in uracil DNA glycosylases repair enzymes removing the deaminated base pairs, and the legitimate ones. So, instead, DNA uses thymidine, which is distinguishable biochemically from uracil by its extra methyl group. This way the cell gains the essential ability to remove the uracils that are a result of deamination using uracil DNA glycosylase enzymes, preventing mass mutation in its genome without removing the thymine base that it actually needs to be there.

The buildup of these “illegitimate” uracils could be catastrophic for the organism - at the very least, copying fidelity of DNA would be detrimentally affected. Thus, cells have repair systems in place to remove these “illegitimate” uracils. But if uracil were already present in DNA, paired to adenine, the repair system would be forced to somehow differentiate between “illegitimate” and “legitimate” uracils. An easy solution to this problem? Add a methyl group to all of the “legitimate” uracils, allowing the repair system to easily tell between the two. This usage of methylated uracil, or thymine, in DNA allowed for the long-term storage of crucial genetic information. 6

In a DNA organism, deoxyuridine can naturally be distinguished from thymidine and be repaired to cytosine. This process cannot take place in the case of RNA deamination, highlighting the great advantage of the invention of thymidine.

But why would prebiotic molecules without distant goals nor purpose to produce a stable information storage medium, DNA, promote this base exchange, and produce error check and repair mechanisms to keep the information intact and promote high-fidelity replication and maintain the mutation levels low ?

Only one extra synthetic step in nucleotide biosynthesis is required to achieve the exchange of uracil to thymine, but the machinery to do the job is enormously complex. 

After further phosphorylation, that is, adding two phosphate groups to the deoxynucleotide monophosphates, they become deoxynucleotide triphosphates dGTP, dATP, dCTP, and dTTP and can be used as the building blocks to construct DNA.

The deoxynucleotide triphosphates (dNTPs) are the building blocks for DNA replication (they lose two of the phosphate groups in the process of incorporation and polymerization). 7  Phosphorylation status of nucleotides is regulated by NDP kinases and NMP kinases that use ATP pool as their cross-phosphorylation source.

Thymidine is a deoxyuridine with a methyl group at the C5 position of the uracil base. This subtle difference plays a critical role in the superior fidelity of DNA-based replication over its RNA counterpart.
Methylation protects the DNA. Beside using thymine instead of uracil, most organisms also use various enzymes to modify DNA after it has been synthesized. Two such enzymes, dam and dcm methylate adenines and cytosines, respectively, along the entire DNA strand. This methylation makes the DNA unrecognizable to many Nucleases (enzymes which break down DNA and RNA), so that it cannot be easily attacked by invaders, like viruses or certain bacteria. Obviously, methylating the nucleotides before they are incorporated ensures that the entire strand of DNA is protected.

Thymine also protects the DNA in another way. If you look at the components of nucleic acids, phosphates, sugars, and bases, you see that they are all very hydrophilic (water soluble). Obviously, adding a hydrophobic (water insoluble) methyl group to part of the DNA is going to change the characteristics of the molecule. The major effect is that the methyl group will be repelled by the rest of the DNA, moving it to a fixed position in the major groove of the helix. This solves an important problem with uracil - though it prefers adenine, uracil can base-pair with almost any other base, including itself, depending on how it situates itself in the helix. By tacking it down to a single conformation, the methyl group restricts uracil (thymine) to pairing only with adenine. This greatly improves the efficiency of DNA replication, by reducing the rate of mismatches, and thus mutations.

To sum up: the replacement of thymine for uracil in DNA protects the DNA from attack and maintains the fidelity of DNA replication.

We will see now, how complex the process and the machinery is, to transform uracil into thymine. This raises the question: How can we best explain the origin of the mechanism of this replacement, which follows a logic and goal, which is a pre-requisite for life to take off?

Synthesis of the Thymine Nucleotides
DNA contains thymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonucleotides. Unlike other deoxynucleotides, thymidylate (2′-deoxythymidine- 5′-monophosphate; dTMP) cannot be directly synthesized by a ribonucleotide reductase, and its de novo biosynthesis requires the enzyme thymidylate synthase. 14  Thymidylate is an essential requirement for deoxyribonucleic acid (DNA) synthesis and normal growth. Uracil, produced by the pyrimidine synthesis pathway, is not a component of DNA. Rather, DNA contains thymine, a methylated analog of uracil.  Another step is required to generate thymidylate from uracil. 

Biosynthesis of thymidylate (dTMP)
The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure above gives details of the thymidylate synthase reaction.

The pathway starts with uridine diphosphate (UDP) to deoxyUridine diphosphate (dUDP) by ribonucleotide reductase. Next, dUDP is converted into deoxyuridine triphosphate ( dUTP ) by the action of a ubiquitous enzyme, nucleoside diphosphate kinase. Next, dUTP is converted to  uridylate (dUMP) by  dUTP phosphatase ( dUTPase). In the next step, dUMP is catalyzed into thymidylate (dTMP) by thymidylate synthase. Next,  dTMP is phosphorylated to form thymidylate triphosphate (dTTP).

Interestingly, formation of uridylate ( dUMP ) from deoxyuridine diphosphate ( dUDP ) passes through uridylate ( dUTP ), which is then cleaved by dUTPase, a pyrophosphatase that removes PPi from dUTP. The action of dUTPase prevents deoxyuridine triphosphate ( dUTP ) from serving as a substrate in DNA synthesis. An alternative route to deoxyuridine monophosphate ( dUMP ) formation starts with deoxycytidine diphosphate (dCDP), which is dephosphorylated to deoxycytidine monophosphate (dCMP) and then deaminated by deoxycytidine monophosphate dCMP deaminase, leaving deoxyuridine monophosphate ( dUMP ).

The apparent reason for the energetically wasteful process of dephosphorylating dUTP and rephosphorylating dTMP is that cells must minimize their concentration of dUTP, and this reaction must be efficient in order to prevent incorporation of uracil into their DNA (the enzyme system that synthesizes DNA from dNTPs does not efficiently discriminate between dUTP and dTTP; dUTPase is a homotrimer. Its X-ray structure reveals the basis for the enzyme’s exquisite specificity for dUTP. Each subunit binds dUTP in a snug-fitting cavity that sterically excludes thymine’s C5 methyl group via the side chains of conserved residues. Thus, dUTPase eliminates dUTP from the DNA biosynthetic pathway. Deficiency or impaired enzymatic activity of dUTPase leads to the elevation of the intracellular dUTP pool and can lead to enhanced uracil incorporation into the DNA. If high genomic uracil content overloads the base-excision repair capacity it will result in DNA fragmentation and subsequent cell death. Proper dUTPase function is thus of key importance in preventing DNA uracilation and hence, in preserving genome integrity. 19

The biosynthesis of thymidine triphosphate is obviously more complex than that of any other dNTP given that thymine nucleotides are derived from uracil nucleotides and a methyl group must be created. In most cells, the dUDP produced by ribonucleotide reductase action on UDP is phosphorylated to dUTP by nucleoside diphosphate kinase. dUTP is cleaved by an active pyrophosphatase, deoxyuridine triphosphatase (dUTPase). This enzyme plays a dual role: (1) Because dUTP is a good substrate for DNA polymerase, dUTPase minimizes the steady-state pool size of dUTP and, hence, helps to exclude dUMP from DNA. (2) The dUTPase reaction is a significant biosynthetic route to dUMP, the substrate for thymidylate synthase. Thymidylate synthase catalyzes the transfer of a single-carbon functional group from 5,10-methylenetetrahydrofolate to position 5 of the pyrimidine ring in deoxyuridine monophosphate, yielding thymidine monophosphate. dTMP is phosphorylated to dTDP by , and conversion to dTTP involves nucleoside diphosphate kinase. 22

dUTPase plays a role in providing dUMP for the thymidylate synthase reaction. Of equal or greater importance is its role in minimizing the incorporation of dUMP into DNA in place of dTMP (see Deoxyuridine Triphosphatase). Cells possess two independent mechanisms to minimize the amount of dUMP in DNA: dUTPase acts to deplete the dUTP pool, and hence to minimize dUMP incorporation by DNA polymerase, usually opposite dAMP in template DNA. The second mechanism is a base excision repair process, starting with . This enzyme scans DNA, and when it contacts a dUMP residue, it cleaves the glycosidic bond, probably by a base-flipping mechanism. This reaction creates an abasic site, which initiates a repair process that results in insertion of the correct nucleotide at that site.

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) 
dUTPase enzymes play an essential role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. 18  dUTP is constantly produced in the pyrimidine biosynthesis network. To prevent uracil incorporation into DNA, representatives of the dUTP  nudeotidehydrolase (dUTPase) enzyme family eliminate excess dUTP.  dUTPases typically possess exquisite specificity and display an intriguing homotrimer active site architecture. Conserved residues from all three monomers contribute to each of the three active sites within the dUTPase. The dUTPase binding pocket is highly specific for uracil.

The dUTPase molecule must be constructed to exacting design specifications in order to cleave efficiently one deoxyribonucleoside triphosphate while having little or no effect on structurally similar dNTPs needed for DNA synthesis, in particular, dUTP and dTTP. Accordingly, kinetic analysis dUTPase showed the ratio kcat/Km to be, remarkably, more than 105-fold lower for dCTP than for dUTP. The nucleotides dTTP and UTP were even poorer substrates as no action of dUTPase on these nucleotides was detectable.

The thymine “invention” required a de novo biosynthesis route. However, mere availability of dTTP is not enough to prevent uracil incorporation into DNA. Most DNA polymerases cannot distinguish between thymine and uracil.  It is the relative level of the respective dNTPs (dUTP/dTTP) that defines incorporation ratios, and therefore, to keep dUMP out of DNA, dUTP levels have to be strictly regulated. The enzyme dUTPase (dUTP nucleotidehydrolase) is responsible for this task: it catalyzes the cleavage of dUTP into dUMP and inorganic pyrophosphate thereby fulfilling a dual role.  On one hand, dUTPase controls dUTP level. On the other hand, the product dUMP is the precursor for dTMP biosynthesis. All free-living organisms and also diverse DNA and RNA (including retro-) viruses encode dUTPase. Although each subunit contains all necessary residues for substrate binding, trimer formation is indispensable to bring these residues in proximity for the cognate binding site. To prevent wasteful hydrolysis of energy-containing NTPs and dNTPs, specificity is of utmost importance for dUTPase. Two major mechanisms provide this: (i) steric exclusion of purines, thymine, and ribose and (ii) a hydrogen bonding pattern specific only for uracil.

The key resolvable enzymatic steps include (i) rapid substrate binding followed by (ii) a relatively slow substrate-induced isomerization to the catalytically competent active site conformation, (iii) the rate-limiting hydrolysis step, and (iv) rapid release of the products.

Thymidylate synthetase
Thymidylate synthase (TS) shares with ribonucleotide reductase the distinction of being responsible for one of the two chemical differences between DNA and RNA. TS generates the methyl group of thymine. This is the only known biological methylation reaction not involving S-adenosylmethionine or the synthesis of methionine itself. 23

Thymidylate synthetase (TS) is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Thymidine is one of the nucleotides in DNA. With inhibition of TS, an imbalance of deoxynucleotides and increased levels of dUMP arise. Both cause DNA damage. 9 Thymidylate synthase is a strictly essential enzyme of DNA precursor biosynthesis. 

This provides the de novo pathway for production of dTMP and is the only enzyme in folate metabolism in which the 5,10-methylenetetrahydrofolate is oxidized during one-carbon transfer. The enzyme is essential for regulating the balanced supply of the 4 DNA precursors in normal DNA replication: defects in the enzyme activity affecting the regulation process cause various biological and genetic abnormalities, such as thymineless death. Thymidylate synthase (TS) plays a crucial role in the early stages of DNA biosynthesis. Synthesis and insertion of healthy DNA is vital for normal body functions and avoidance of cancerous activity. In addition, inhibition in synthesis of important nucleotides necessary for cell growth is important.

Crystal structure of Thymidylate synthetase 

Thymidylate synthase mechanism and reaction
In the thymidylate synthase reaction, the transferred methylene group must be reduced to the methyl level, and the electron pair that brings this reduction about comes from the reduced pteridine ring of 5,10-methylenetetrahydrofolate. The coenzyme, therefore, loses both its methylene group and an electron pair, leading to dihydrofolate. Transformation of the coenzyme for reuse involves, first, its reduction to tetrahydrofolate by dihydrofolate reductase and, next, transfer of a single-carbon group to the pteridine ring, usually catalyzed by serine transhydroxymethylase. . Such inhibitors include Methotrexate, widely used in cancer chemotherapy, and Trimethoprim, an antibacterial agent that specifically inhibits dihydrofolate reductases of prokaryotic origin(see Aminopterin). 

TS forms a covalent bond to the substrate dUMP through a 1,4-addition involving a cysteine nucleophile. The coenzyme tetrahydrofolate donates a methyl group to the alpha carbon while reducing the new methyl on dUMP to form dTMP.

Thymidylate synthase catalyzes this finishing touch: deoxyuridylate (dUMP) is methylated to thymidylate (TMP). The methyl donor in this reaction is N5,N10 -methylenetetrahydrofolate rather than S-adenosylmethionine 8 The methyl group becomes attached to the C-5 atom within the aromatic ring of dUMP, but this carbon atom is not a good nucleophile and cannot itself attack the appropriate group on the methyl donor. Thymidylate synthase promotes methylation by adding a thiolate from a cysteine side chain to this ring to generate a nucleophilic species that can attack the methylene group of N5, N10-methylenetetrahydrofolate (Figure below).

Thymidylate synthesis.
Thymidylate synthase catalyzes the addition of a methyl group (derived from N5, N10- methylenetetrahydrofolate) to dUMP to form TMP. The addition of a thiolate from the enzyme activates dUMP. Opening the five-membered ring of the THF derivative prepares the methylene group for nucleophilic attack by the activated dUMP. The reaction is completed by the transfer of a hydride ion to form dihydrofolate.

This methylene group, in turn, is activated by distortions imposed by the enzyme that favor opening the five-membered ring. The activated dUMP’s attack on the methylene group forms the new carbon–carbon bond. The intermediate formed is then converted into product: a hydride ion is transferred from the tetrahydrofolate ring to transform the methylene group into a methyl group, and a proton is abstracted from the carbon atom bearing the
methyl group to eliminate the cysteine and regenerate the aromatic ring. The tetrahydrofolate derivative loses both its methylene group and a hydride ion and, hence, is oxidized to dihydrofolate. For the synthesis of more thymidylate, tetrahydrofolate must be regenerated.

The unique property of the action of thymidylate synthetase is that the N5,N10-methylene THF is converted to dihydrofolate (DHF), the only such reaction yielding DHF from THF. In order for the thymidylate synthetase reaction to continue, THF must be regenerated from DHF. This is accomplished through the action of dihydrofolate reductase (DHFR). THF is then converted to N5,N10-THF via the action of serine hydroxymethyltransferase.

The three enzymes of the thymidylate synthesis cycle are:

serine hydroxymethyltransferase (SHMT)
thymidylate synthase (TS)
dihydrofolate reductase (DHFR)

5,10-Methylenetetrahydrofolate is the substrate for the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP).

Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase 
Serine hydroxymethyltransferase is required for regeneration of the N5, N10-methylene form of tetrahydrofolate. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5, N10-methylenetetrahydrofolate(pink and gray)

Conversion of dUMP to dTMP and its inhibition by FdUMP. 
The top row is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton in step 3 . The hydrogens derived from the methylene group of N5, N10-methylenetetrahydrofolate are shaded in gray. A novel feature of this reaction mechanism is a 1,3 hydride shift (step 3 ), which moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. It is this hydride shift that apparently does not occur when FdUMP is the substrate (bottom). Steps 1 and 2 proceed normally, but result in a stable complex— with FdUMP linked covalently to the enzyme and to tetrahydrofolate—that inactivates the enzyme.

Thymidylate synthase, a highly conserved  homodimeric protein, proceeds upon following mechanistic scheme: 

Catalytic mechanism of thymidylate synthase 
The methyl group is supplied by N5,N10-methylene-THF, which is concomitantly oxidized to dihydrofolate.

1. An enzyme nucleophile, identified as the thiolate group of Cys 146, attacks C6 of dUMP to form a covalent adduct.
2. C5 of the resulting enolate ion attacks the CH2 group of the iminium cation in equilibrium with N5,N10-methylene-THF to form an enzyme– dUMP–THF ternary covalent complex.
3. An enzyme base abstracts the acidic proton at the C5 position of the enzyme-bound dUMP, forming an exocyclic methylene group and eliminating the THF cofactor. The abstracted proton subsequently exchanges with solvent.
4. The redox change occurs via the migration of the C6-H atom of THF as a hydride ion to the exocyclic methylene group, converting it to a methyl group and yielding DHF. This reduction promotes the displacement of the Cys thiolate group from the intermediate to release product, dTMP, and re-form the active enzyme.

Tetrahydrofolate Is Regenerated in Two Reactions
The thymidylate synthase reaction is biochemically unique in that it oxidizes THF to DHF; no other enzymatic reaction employing a THF cofactor alters this coenzyme’s net oxidation state. The DHF product of the thymidylate synthase reaction is recycled back to N5,N10-methylene-THF through two sequential reactions: 

Regeneration of N5,N10- methylenetetrahydrofolate. 
The DHF product of the thymidylate synthase reaction is converted back to N5,N10-methylene-THF by the sequential actions of (1) dihydrofolate reductase and (2) serine hydroxymethyltransferase. The sites of action of some inhibitors are indicated by red octagons. Thymidylate synthase is inhibited by FdUMP, whereas dihydrofolate reductase is inhibited by the antifolates methotrexate, aminopterin, and trimethoprim

1. DHF is reduced to THF by NADPH as catalyzed by dihydrofolate reductase. Although in most organisms DHFR is a monomeric, monofunctional enzyme, in protozoa and some plants DHFR and thymidylate synthase occur on the same polypeptide chain to form a bifunctional enzyme that has been shown to channel DHF from its thymidylate synthase to its DHFR active sites.
2. Serine hydroxymethyltransferase transfers the hydroxymethyl group of serine to THF yielding N5,N10-methylene-THF and glycine.

Dihydrofolate reductase catalyzes the regeneration of tetrahydrofolate, a one-carbon carrier
Tetrahydrofolate is regenerated from the dihydrofolate that is produced in the synthesis of thymidylate. This regeneration is accomplished by dihydrofolate reductase with the use of NADPH as the reductant. ( see figure below )

DNA contains thymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonucleotides. The immediate precursor of thymidylate (dTMP) is dUMP. In bacteria, the pathway to dUMP begins with formation of dUTP, either by deamination of dCTP or by phosphorylation of dUDP

Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase (Figure below). This enzyme methylates dUMP at the 5-position to create dTMP; the methyl donor is the one-carbon folic acid derivative N5,N10-methylene-THF. The reaction is actually a reductive methylation in which the one-carbon unit is transferred at the methylene level of reduction and then reduced to the methyl level.

(a) The thymidylate synthase reaction. The 5-CH3 group is ultimately derived from the b-carbon of serine. 
(b) Thymidylate synthase dimer. Each monomer has a bound folate analog (green) and dUMP (light blue)

The THF cofactor is oxidized at the expense of methylene reduction to yield DHF. DHFR then reduces DHF back to THF for service again as a one-carbon vehicle. Thymidylate synthase sits at a junction connecting dNTP synthesis with folate metabolism. Purine synthesis is affected as well because it is also dependent on THF.

Dihydrofolate reductase

Crystal structure of dihydrofolate reductase.

Dihydrofolate reductase, or DHFR, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry.  Found in all organisms, DHFR has a critical role in regulating the amount of tetrahydrofolate in the cell. Tetrahydrofolate and its derivatives are essential for purine and thymidylate synthesis, which are important for cell proliferation and cell growth. DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate.[14] In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to NADP+16

Serine hydroxymethyltransferase

Crystal structure of serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a Pyridoxal phosphate (PLP) (Vitamin B6) dependent enzyme which plays an important role in cellular one-carbon pathways by catalyzing the reversible, simultaneous conversions of L-serine to glycine and tetrahydrofolate (THF) to 5,10-methylenetetrahydrofolate (5,10-CH2-THF). This reaction provides the largest part of the one-carbon units available to the cell. 17

An Alternative Flavin-Dependent Mechanism for Thymidylate Synthesis
For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. Thymidine is unique to DNA can be synthesized by two radically
different enzymes that have no similarity in sequence or structure. This reaction catalyzed thymidylate synthases (thyA) is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. 12 Certain bacteriological and archeological organisms have been found to lack any gene coding for such TSase, as well as dihydrofolate reductase and thymidine kinase, yet can survive in thymidine-deprived environments. This observation led to the identification of alternative flavin-dependent thymidylate synthases 14

Thymidylate (deoxythymidine 5′-monophosphate or dTMP) is a key metabolite required for the accurate replication of DNA genomes in all cellular organisms. Until recently, thymidylate synthase ThyA was thought to correspond to the sole enzyme catalyzing the formation of dTMP de novo. However, recently a family of thymidylate synthases (ThyXs) was identified. 

Flavin-dependent thymidylate synthase tetramer  13
Most organisms, including humans, use the thyA- or TYMS-encoded classic thymidylate synthase whereas some bacteria use the similar flavin-dependent thymidylate synthase (FDTS) instead

The residues essential for catalysis in ThyA are absent in ThyX proteins. 10 Multiple studies have identified key differences in the molecular mechanism of catalysis between flavin dependent TSs ( FDTS's) and classic TSases. 

This is remarkable. An enzyme, that has no homology, that is, no shared ancestry, and had to emerge prior when life began, that means the prebiotic convergent emergence of an enzyme with the same function and reaction, but it emerged twice by different routes.  That should be perplexing for any advocate of naturalistic explanations.

Although deoxythymidylate cannot be provided directly by ribonucleotide reductase, the gene encoding thymidylate synthase ThyA is absent from the genomes of a large number of nonsymbiotic microbes. We show that ThyX  proteins of previously unknown function form a large and distinct class of thymidylate synthases. ThyX has a wide but sporadic phylogenetic distribution, almost exclusively limited to microbial genomes lacking thyA. ThyX and ThyA use different reductive mechanisms because ThyX activity is dependent on reduced flavin nucleotides. Our findings reveal complexity in the evolution of thymidine in present-day DNA. 11

It is common that science papers do not distinguish between enzymes, that had to emerge prebiotically, prior when life began, and enzymes that came after a supposed minimal LUCA. It is here the case, where they mention evolution, despite the fact that evolution depends on DNA. And DNA depends on these enzymes to synthesize (dTMP).

All deoxythymidine 5'monophosphate (dTMP) in bacteria and eukarya is thought to be formed either de novo by thymidylate synthase (ThyA)–dependent methylation of deoxyuridine 5-monophosphate (dUMP) or by thymidine kinase (Tdk)–dependent salvage of thymidine compounds from the growth medium. ThyA uses tetrahydrofolate (H4folate) as a reductant and forms dihydrofolate (H2folate) as a product of the methylation reaction. Because reduced folate derivatives are essential for a variety of biological processes, H2folate formed by ThyA is rapidly reduced to H4folate by dihydrofolate reductase (DHFR). This functional, and often physical, coupling of ThyA and DHFR proteins was thought to be essential for de novo thymidylate synthesis in virtually all actively dividing cells.

thyX and thyA genes have mutually exclusive phylogenetic patterns . On the basis of which we predict that ThyX has substituted for the unrelated ThyA protein.  thyX can functionally replace thyA in dTMP synthesis.

FDTS requires a nicotinamide cofactor to reduce the bound flavin. FDTS enzymes use a flavin adenine dinucleotide (FAD) prosthetic group to catalyze the redox chemistry, which can be divided into reductive and oxidative-half reactions. The FAD can be reduced to FADH2 by nicotinamides, ferrodoxin, and other small molecule reductants (e.g., dithionite). Although the reductive half-reaction is activated by dUMP the conversion of dUMP to dTMP occurs during the oxidative half-reaction (FADH+ →FAD)  In contrast to classic TSase, where the cofactor CH2H4 folate provides both the H− and methylene, in the FDTS reaction the H− is provided by the FADH+ and CH2H4 folate is used only as a source for the methylene moiety. 

Mechanisms for FDTS catalyzed methylene transfer. 
(A) A recently proposed mechanism for FDTS enzymes involving direct transfer of the methylene between CH2H4folate and dUMP. 
(B) A mechanism proposed for methylene transfer that involves an enzymatic arginine residue. Please note that the methylene transfer is the focus of this figure, and the methylene could in principle be transfered to the reduced dUMP (as drawn), to the oxidized dUMP as proposed in ref. 8, or to dUMP activated by Michael addition of Ser to C6, as originally suggested by ref. 16. R, 2′-deoxyribose-5′-phosphate; R′, (p-aminobenzoyl)-glutamate.

The mechanism of the FDTS-catalyzed reaction differs greatly from that of the classical thymidylate synthases. Perhaps the most notable distinction between these pathways toward thymidylate, is the role of an enzymatic nucleophile. Classical thymidylate synthases absolutely require an active site nucleophile to covalently activate the substrate, dUMP. FDTSs, on the other hand, lack such functionality leading to a chemical cascade where the dUMP and intermediates along the reaction path, do not covalently bind the enzyme. Instead, the FDTS-catalyzed reaction relies on the reduced flavin cofactor, which has been suggested to reduce the uracil moiety by hydride transfer.

Evolution of Thymidylate Synthase
In the paper: An Evolutionary Analysis of Lateral Gene Transfer in Thymidylate Synthase Enzymes, the authors write as follows:
Thymidylate synthases are essential for all DNA-based forms of life and therefore implicated in the hypothesized transition from RNA genomes to DNA genomes. Two evolutionally unrelated Thy enzymes, ThyA and ThyX, are known to catalyze the same biochemical reaction. For decades, only one family of thymidylate synthase enzymes was known, and its presence was considered necessary to maintain all DNA-based forms of life. Then, a gene encoding an alternative enzyme was discovered and characterized. The novel enzyme was named ThyX, whereas the other enzyme was renamed ThyA. The 2 enzymes, ThyA and ThyX, were found to have distinctly different sequences and structures, thus alluding to independent evolutionary origins.

It is remarkable that most, if not all scientific papers, do not make a distinction between life essential parts that had to emerge prior life started, and other parts that came afterward. A clear distinction should be made what can be explained by biological evolution, and what cannot. Thymidylate Synthase is claimed to be necessary in a transition state from the so-called RNA to the DNA world. The article: Ancient enzymes reveal the DNA genesis says as follows: " Nature made at least three new types of inventions in assembling living cells from building blocks produced by prebiotic chemistry", and continues: " heritable blueprints – genetic coding –furnished sufficient continuity for complexity to grow. The most dramatic of these inventions were all completed and probably overwritten before the first living cells appeared.  So, DNA is clearly a pre-life invention. And cannot be explained by Darwinian evolution.

By virtue of their function and phyletic distribution, Thys are ancient enzymes, implying 1) the likely participation of one or both enzymes during the transition from an RNA world to a DNA world (based on protein catalysts: and 2) the probable presence of a gene encoding Thy in the genome of the common ancestors of eukaryotes, bacteria, and archaea.

This claim is even more remarkable. The authors suggest a pre-existing gene encoding these enzymes. How could that be, if the enzyme is responsible to produce thymidylate triphosphate (dTTP), one of the four nucleotides that constitute DNA? - and therefore, genes were not existing yet? This is one of the many classic chicken-egg situations, which are encountered all over in biological Cells. 

"The evolutionary phenomenon whereby the same archaeon or bacterium encodes 2 structurally distinct enzymes performing the same function is postulated to be a possible transition stage in lateral gene transfer (LGT) and subsequent gene loss".

This is a remarkable ad-hoc claim. The authors do not question, why a prebiotic, pre-life mechanism, be it chemical evolution, self-assembly, physical laws or mere chance would have come up with two distinct complex mechanisms, performing the same reaction. These questions would not find a satisfying answer based on naturalism. So wipe them under the table and not asking, is more comfortable. But that does not put them away. There is no way these enzymes could have emerged prebiotically, by mere naturalistic means, and then, the authors make up another just so scenario: The two routes are explained by lateral gene transfer. No more, no less. This is nothing more than inventing stories. It has nothing to do with science and makes no sense whatsoever.

But there is more. How could lateral gene transfer explain the makeup of the second enzyme, if it is not homologous?  - that is if the amino acid sequence is largely different? 

a  Biological hydrolysis is the cleavage of biomolecules where a water molecule is consumed to effect the separation of a larger molecule into component parts. 1

b In biochemistry and molecular genetics, an AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA (also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage. It has been estimated that under physiological conditions 10,000 apurinic sites and 500 apyrimidinic may be generated in a cell daily. 4

The resulting abasic site is then recognized by AP endonuclease enzymes that break a phosphodiester bond in the DNA, permitting the repair of the resulting lesion by replacement with another cytosine. A DNA polymerase may perform this replacement via nick translation, a terminal excision reaction by it's 5'-->3' exonuclease activity, followed by a fill-in reaction by its polymerase activity. DNA ligase then forms a phosphodiester bond to seal the resulting nicked duplex product, which now includes a new, correct cytosine. 

5. Molecular biology of the Cell, 6th ed. page 268     
8. Biochemistry , 8th ed. Styer, page 755
15. Fundamentals of biochemistry, 5th ed. page 818

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9 Re: The DNA double helix - evidence of design on Thu Oct 26, 2017 8:08 am



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10 Prebiotic Nucleic Acid synthesis on Sat Nov 18, 2017 9:52 am


Prebiotic Nucleic Acid synthesis

The same year that Miller published his pioneering result regarding abiotic amino acid synthesis, the double-helical model for the structure of DNA was published (Watson and Crick 1953), which effectively clinched the role of nucleic acids in the inheritance of biological mutations. There are two types of nucleic acids important in biological systems, DNA and RNA, linked by the processes of transcription and biosynthesis
(Figure 5.1).

Not long after Miller’s and Watson and Crick’s discoveries, the search for abiotic mechanisms for the synthesis of these important biochemicals began. Oró and Kimball showed that adenine, a biological purine, could be derived from the polymerization of aqueous HCN, of which it is formally a pentamer (C5H5N5) (Oró and Kimball 1961). The mechanism of this synthesis was soon elucidated, and shortly thereafter, syntheses of other important purines including guanine, and the biosynthetic precursor's xanthine and hypoxanthine were elaborated (Ferris and Orgel 1965, 1966) (Figure 5.11).

Divergent prebiotic synthesis of pyrimidine and 8-oxo-purine ribonucleotides 19
Understanding prebiotic nucleotide synthesis is a long-standing challenge thought to be essential to elucidate the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Our results suggest that further investigation of the informational and functional properties of the 8-oxo-purine ribonucleotides is warranted.

Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions 20
At some stage in the origin of life, an informational polymer must have arisen by purely chemical means. According to one version of the ‘RNA world’ hypothesis this polymer was RNA, but attempts to provide experimental support for this have failed. In particular, although there has been some success demonstrating that ‘activated’ ribonucleotides can polymerize to form RNA, it is far from obvious how such ribonucleotides could have formed from their constituent parts (ribose and nucleobases). Ribose is difficult to form selectively, and the addition of nucleobases to ribose is inefficient in the case of purines and does not occur at all in the case of the canonical pyrimidines. Although the issue of temporally separated supplies of glycolaldehyde and glyceraldehyde remains a problem, a number of situations could have arisen that would result in the conditions of heating and progressive dehydration followed by cooling, rehydration and ultraviolet irradiation.

Cytosine  is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA).

Dozens of enzymes are needed to make the DNA bases cytosine and thymine from their component atoms. 5 The first step is a "condensation" reaction, connecting two short molecules to form one longer chain, performed by  Aspartate carbamoyltransferase. Other enzymes then connect the ends of this chain to form the six-sided ring of nucleotide bases, and half a dozen others shuffle atoms around to form each of the bases.

In bacteria, the first enzyme in the sequence, aspartate carbamoyltransferase, controls the entire pathway. (In human cells, the regulation is more complex, involving the interaction of several of the enzymes in the pathway.) Bacterial aspartate carbamoyltransferase determines when thymine and cytosine will be made, through a battle of opposing forces. It is an allosteric enzyme, referring to its remarkable changes in shape (the term is derived from the Greek for "other shape"). The enzyme is composed of six large catalytic subunits and six smaller regulatory subunits.

The active site of the enzyme is located where two individual catalytic subunits touch, so the position of the two subunits relative to one another is critical. If the two subunits are in tight contact, an amino acid from one extends into the active site of the other, blocking its action. If the two are pulled slightly apart, however, the active sites are revealed, allowing molecules to enter and the reaction to be performed. This is the job of the regulatory subunits: they alternately pull the central catalytic subunits apart, turning the enzyme on, or allow them to stick together,  turning the entire complex off. 

Take just a moment to ponder the immensity of this enzyme. The entire complex is composed of over 40,000 atoms, each of which plays a vital role. The handful of atoms that actually perform the chemical reaction are the central players. But they are not the only important atoms within the enzyme--every atom plays a supporting part. The atoms lining the surfaces between subunits are chosen to complement one another exactly, to orchestrate
the shifting regulatory motions. The atoms covering the surface are carefully picked to interact optimally with water, ensuring that the enzyme doesn't form a pasty aggregate, but remains an individual, floating factory. And the thousands of interior atoms are chosen to fit like a jigsaw puzzle, interlocking into a sturdy framework. Aspartate carbamoyltransferase is fully as complex as any fine automobile in our familiar world. And, just as manufacturers invest a great deal of research and time into the design of an automobile, enzymes like aspartate carbamoyltransferase are finely tuned.

Prebiotic synthesis
Regardless, a few assumptions on substrate availability in the prebiotic earth, have led to questions of what is possible in terms of generating usable building blocks leading to nucleic acids. Most studies have focused on:

(1) the prebiotic synthesis of the pyrimidine and purine bases;
(2) the synthesis of ribose;
(3) connecting the two to make nucleosides and nucleotides; and, lastly,
(4) the formation of long enough polymers to give rise to catalytically active RNA and finally tRNAs  8

1. ASTROBIOLOGY An Evolutionary Approach, page 103

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11 Re: The DNA double helix - evidence of design on Tue Jun 19, 2018 7:58 pm


DNA synthesis - what came first, the enzymes to make DNA, or DNA to make the enzymes that synthesize DNA?

These are some enzyme names of de novo Purine and Pyrimidine biosynthesis, essential to make DNA, and supposedly extant at LUCA ( last universal common ancestor )

to make purines:

The enzymes of de novo purine synthesis

Phosphoribosyl-pyrophosphate synthetase (Prs) 

1. Ribose-phosphate diphosphokinase
2. amidophosphoribosyl transferase
3. Phosphoribosylglycinamide formyltransferase ( GAR )
4. Phosphoribosylaminoimidazole carboxylase
5. Dihydrofolate reductase
6. AIR synthetase
7. AIR carboxylase
8. SAICAR synthetase
9. adenylosuccinase (adenylosuccinate lyase)
10. AICAR transformylase
11. IMP cyclohydrolase 

The enzymes for Adenine synthesis

1. Adenylosuccinate synthase 
2. adenylosuccinase (adenylosuccinate lyase)

The enzymes for Guanine synthesis

1. IMP dehydrogenase
2. GMP synthase 

Enzymes used to make Pyrimidines

1. Carbamoyl phosphate synthase II
2. Aspartate carbamoyltransferase
3. Dihydroorotase
4. Dihydro Orotate Dehydrogenase
5. Orotate Phosphoribosyl transferase
6. Orotidine 5'-phosphate decarboxylase
7. Nucleoside-phosphate kinase  & Nucleoside-diphosphate kinase

How did these enzymes emerge on a prebiotic earth ???

It takes DNA to make these enzymes. And it takes these enzymes to make DNA.......

But there is more.

What came first, ATP or the enzymes that use ATP, to make ATP ?

1. ATP drives proteins that make Adenosine monophosphate ( AMP ). 
2. ATP drives enzymes that make Adenosine diphosphate (ADP). 
3. ATP drives enzymes that make Adenosine triphosphate (ATP). ==>> return to 1.
1. ATP drives proteins that make Adenosine monophosphate ( AMP ). 
2. ATP drives enzymes that make Adenosine diphosphate (ADP). 
3. ATP drives enzymes that make Adenosine triphosphate (ATP).
====>>> endless loop.

The Adenine triphosphate (ATP) molecule as energy source is required to drive the enzymes/protein machines that make the adenine nucleic base and adenosine monophosphate AMP, used in DNA, one of the four genetic nucleotides "letters" to write the Genetic Code, and then, using these nucleotides as starting material, then further molecular machines attach other two phosphates and produce adenine triphosphates (ATP) - the very own molecule which is used as energy source to drive the whole process.. What came first: the enzymes to make ATP, or ATP to make the enzymes that make ATP?

But lets suppose abiogenesis would produce RNA on a prebiotic earth, and so, AMP, ADP, and ATP. How could there be a transition of monomers to polyperisation ?


The RNA world hypothesis, to be true, has to overcome  major hurdles:

1. Life uses only right-handed RNA and DNA. The homochirality problem is unsolved. This is an “intractable problem” for chemical evolution
2. RNA has been called a “prebiotic chemist's nightmare” because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic instability. Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a “water problem”. Finally, some bonds in RNA appear to be “impossible” to form under any conditions considered plausible for early Earth.   In chemistry, when free energy is applied to organic matter without Darwinian evolution, the matter devolves to become more and more “asphaltic”, as the atoms in the mixture are rearranged to give ever more molecular species. In the resulting “asphaltization”, what was life comes to display fewer and fewer characteristics of life.
3. Systems of interconnected software and hardware like in the cell are irreducibly complex and interdependent. There is no reason for information processing machinery to exist without the software and vice versa.
4. A certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded
5.  RNA catalysts would have had to copy multiple sets of RNA blueprints nearly as accurately as do modern-day enzymes
6.  In order a molecule to be a self-replicator, it has to be a homopolymer, of which the backbone must have the same repetitive units; they must be identical. On the prebiotic world, the generation of a homopolymer was however impossible.
7. Not one self-replicating RNA has emerged to date from quadrillions (10^24) of artificially synthesized, random RNA sequences.  
8. Over time, organic molecules break apart as fast as they form
9. How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity? And how would non-guided random events be able to attach the nucleic bases to the ribose?  The coupling of a ribose with a nucleotide is the first step to form RNA, and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines.”
10.  L. E. Orgel:  The myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA's catalytic potential.
11. Macromolecules do not spontaneously combine to form macromolecules
125. The transition from RNA to DNA is an unsolved problem. 
13. To go from a self-replicating RNA molecule to a self-replicating cell is like to go from a house building block to a fully build house. 
14. If two amino acids are located within the peptidyl transferase center, they will easily form a peptide bond. But as soon as you do that in the absence of the ribosome, the ends of the amino acids come together, forming a cyclic structure. Polymers cannot form. But if the ends are kept apart, by a theoretical primitive ribosome, a chain of peptide bonds could grow into a polymer. 30
15. Arguably one of the most outstanding problems in understanding the progress of early life is the transition from the RNA world to the modern protein based world. 31 
16. It is thought that the boron minerals needed to form RNA from pre-biotic soups were not available on early Earth in sufficient quantity, and the molybdenum minerals were not available in the correct chemical form. 33

Proponents of the RNA world hypothesis commonly argue that it has been proven that RNA's could self-replicate. Let's suppose that were true, that is as if self-replication could produce a hard drive. To go from a hard drive ( which by itself requires complex information to be assembled, in case of biology, DNA, not RNA since it's too unstable, ) that does not explain the origin of the information to make all life essential parts in the cell.
It is as to go just from a hard drive storage device to a self replicating factory with the ability of self replication of the entire factory once ready, to respond to changing environmental demands and regulate its metabolic pathways, regulate and coordinate all cellular processes, such as molecule and building block biosynthesis according to the cells demands, depending on growth, and other factors.
The ability of uptake of nutrients, to be structured, internally compartmentalized and organized, being able to check replication errors and minimize them, and react to stimuli, and changing environments. That's is, the ability to adapt to the environment is a must right from the beginning. If just ONE single protein or enzyme - of many - is missing, no life. If topoisomerase II or helicase are missing - no replication - no perpetuation of life.
Why would a prebiotic soup or hydrothermal vents produce these proteins - if by their own there is no use for them?

chemist Wilhelm Huck, professor at Radboud University Nijmegen
A working cell is more than the sum of its parts. "A functioning cell must be entirely correct at once, in all its complexity

The cell is irreducibly complex

ATP: The  Energy  Currency for the Cell

Purines and their synthesis

Biosynthesis of the DNA double helix, evidence of design


Biosynthesis of the DNA double helix, evidence of design

DNA is crucial for life. Not many however grasp how complex it is for cells to synthesize DNA molecules. To make Purines it takes a biosynthesis pathway of five enzymes, and Pyrimidines, seven.
Now let's have a closer look at just one of the enzymes to make pyrimidines, the first in the pathway, namely  Aspartate Carbamoyltransferase.

David Goodsell writes in his book: Our molecular nature, on page 26:

Dozens of enzymes are needed to make the DNA bases cytosine and thymine from their component atoms. The first step is performed by aspartate carbamoyltransferase.  In bacteria, this enzyme controls the entire pathway. (In human cells, the regulation is more complex, involving the interaction of several of the enzymes in the pathway.) The enzyme is composed of six large catalytic subunits and six smaller regulatory subunits. The active site of the enzyme is located where two individual catalytic subunits touch, so the position of the two subunits relative to one another is critical. Take just a moment to ponder the immensity of this enzyme. The entire complex is composed of over 40,000 atoms, each of which plays a vital role. The handful of atoms that actually perform the chemical reaction are the central players. But they are not the only important atoms within the enzyme--every atom plays a supporting pan. The atoms lining the surfaces between subunits are chosen to complement one another exactly, to orchestrate the shifting regulatory motions. The atoms covering the surface are carefully picked to interact optimally with water, ensuring that the enzyme doesn't form a pasty aggregate, but remains an individual, noating factory. And the thousands of interior atoms are chosen to fit like a jigsaw puzzle, interlocking into a sturdy framework. Aspartate carbamoyltransferase is fully as complex as any fine automobile in our familiar world.

This is the description of just ONE enzyme.

Now consider that : A minimal estimate for the gene content of the last universal common ancestor
19 December 2005
A truly minimal estimate of the gene content of the last universal common ancestor, obtained by three different tree construction methods and the inclusion or not of eukaryotes (in total, there are 669 ortholog families distributed in 561 functional annotation descriptions, including 52 which remain uncharacterized).

This means, and least 561 protein subunits, cofactors, apo-proteins and protein complexes are required to get a minimal proteome and functioning cell. Most of these proteins are more complex than the described above, and true factories in their own rights, such as the Ribosome, described as one of the most complex proteins known, and crucial for protein synthesis.

We know empirically, that intelligence can and does invent, elaborates, projects, and makes blueprints of complex machines, production lines, and factories, and is capable of implementing them. We do have no example of any alternative causal mechanism able of the same feat. Denton describes biological cells as " veritable micro-miniaturized factories containing thousands of exquisitely designed pieces of intricate molecular machinery, made up altogether of one hundred thousand million atoms, far more complicated than any machine built by man and absolutely without parallel in the non-living world ".

Biosynthesis of the DNA double helix, evidence of design

LUCA—The Last Universal Common Ancestor
The last universal common ancestor represents the primordial cellular organism from which diversified life was derived

The Cell is  a Factory

The possible mechanisms to explain the origin of life

Neither Evolution nor physical necessity are a driving force prior dna replication. The only two alternatives to explain the origin of life, and biological cells,  are either 

a) creation by an intelligent agency, or 
b) Random, unguided, undirected natural events by a lucky "accident".


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12 Re: The DNA double helix - evidence of design on Tue Jun 26, 2018 5:03 pm


Polymeryzation of Phosphodiester bonds

Activated monomers are essential because polymerization reactions occur in an aqueous medium and are therefore energetically uphill in the absence of activation. 4
A central problem, therefore, concerns mechanisms by which prebiotic monomers could have been activated to assemble into polymers.

In life today, the removal of water is performed upstream of the actual bond formation. This process involves the energetically downhill transfer of electrons, which is coupled to either substrate-level oxidation or generation of a proton gradient that in turn is the energy source for the synthesis of anhydride pyrophosphate bonds in ATP. The energy stored in the pyrophosphate bond is then distributed throughout the cell to drive most other energy‐dependent reactions. This is a complex and highly evolved process, so here we consider simpler ways in which energy could have been captured from the environment and made available for primitive versions of metabolism and polymer synthesis. Because the atmosphere of the primitive Earth did not contain appreciable oxygen, this review of primitive bioenergetics is limited to anaerobic sources of energy.

a plausible energy source for polymerization remains an open question. Condensation reactions driven by cycles of anhydrous conditions and hydration would seem to be one obvious possibility but seem limited by the lack of specificity of the chemical bonds that are formed.

Initial studies established that some montmorillonite clays catalyze condensation of activated mononucleotides to oligomers which contain about ten monomer units. For example, the self-condensation of the 5'-phosphorimidazolide of adenosine (ImpA) in pH 8 aqueous solution in the presence of montmorillonite results in the formation of 2-10 mers in which 66% of the phosphodiester bonds are 3',5'-linked.  5

Take the clay used in the Ferris et al. experiments, for instance. Montmorillonite (often used in cat litter) is a layered clay "rich in silicate and aluminum oxide bonds" (Shapiro 2006, 108). But the montmorillonite employed in the Ferris et al. experiments is not a naturally-occuring material, as Ertem (2004) explains in detail. Natural or native clays don't work, because they contain metal cations that interfere with phosphorylation reactions:

(Shapiro 2006, 108)

This handicap was overcome in the synthetic experiments by titrating the clays to a monoionic form, generally sodium, before they were used. Even after this step, the activity of the montmorillionite depended strongly on its physical source, with samples from Wyoming yielding the best results....Eventually the experimenters settled on Volclay, a commercially processed Wyoming montmorillonite provided by the American Colloid Company. Further purification steps were applied to obtain the catalyst used for the "prebiotic" formation of RNA.

Phosphodiester bonds are central to most life on Earth, as they make up the backbone of the strands of DNA. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA. Strong covalent bonds form between the phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds. 1

In order for the phosphodiester bond to be formed and the nucleotides to be joined, the tri-phosphate or di-phosphate forms of the nucleotide building blocks are broken apart to give off energy required to drive the enzyme-catalyzed reaction. When a single phosphate or two phosphates known as pyrophosphates break away and catalyze the reaction, the phosphodiester bond is formed.

Hydrolysis of phosphodiester bonds can be catalyzed by the action of phosphodiesterases which play an important role in repairing DNA sequences.

A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. An example is found in the linking of two pentose (5 carbon sugar) rings to a phosphate group by strong, covalent ester bonds. Each ester bond is formed by a condensation reaction in which water is lost. This bond is a key structural feature of the backbone of DNA and RNA and links the 3’ carbon of one nucleotide to the 5’ carbon of another to produce the strands of DNA and RNA.  2

gif hosting

Ligase is a type of enzyme that forms a link between carbon and either: carbon, sulphur, oxygen, or nitrogen; using the hydrolysis of ATP and its high energy bond to drive formation of the new covelant bond [1]. There are various types of ligases; one of the most important ones is DNA ligase which joins DNA fragments via phosphodiester bonds and is used in processes such as DNA replication where Okazaki fragments need to be annealed in order to complete the formation of the lagging strand.

DNA ligase is a crucial element in recombinant technology 3

No DNA ligase enzymes, no formation of DNA strands, no life. Observe the nomenclature : " recombinant technology".....  

In molecular biology, DNA ligase is a specific type of enzyme, a ligase, (EC that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template,[1] with DNA ligase creating the final phosphodiester bond to fully repair the DNA.

DNA ligase has applications in both DNA repair and DNA replication


6) file:///E:/Downloads/challenges-05-00193%20(3).pdf

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Genetic Molecule formation on a prebiotic earth

The overall scheme for the prebiotic synthesis of a genetic molecule is based on the supposition that organic molecules were available in the environment. . Figure below broadly diagrams the steps that supposedly were involved in the prebiotic synthesis of a primordial genetic polymer. 3

General scheme for the prebiotic origin of a genetic polymer.

Briefly, and importantly, it is likely that given a sufficiently reducing environment, a variety of energetic processes can induce carbon and nitrogen-containing gases to form reactive molecules such as formaldehyde HCHO a,  nitrongen NH3, and HCN b . These can in turn further react, once delivered into an aqueous environment, to form more complex organic compounds such as amino acids, sugars, and heterocyclic compounds. Although the number of possible products grows exponentially as the number of atoms in a compound increases, prebiotic reactions are mechanistically constrained and tend to form a relatively small number of the possible low-molecular-weight organic compounds. Most other sugars and sugar phosphates have half-lives within an order of magnitude of this, thus sugars may be too unstable to have accumulated to signifi cant concentrations in prebiotic environments. These stability estimates do not take into account the competing degradation reactions caused by congener amines, such as ammonia and amino acids. 

While the polymerization of formaldehyde produces ribose, it requires high pH and possibly unreasonably high concentrations of formaldehyde, yields a complex product mixture, and the synthetic conditions also catalyze
the destruction of the products. Sugars are also notoriously unstable; for example, at pH 7 and 25°C, the half-life for ribose decomposition is estimated at 300 days. 

Thus far, the demonstrated prebiotic syntheses of purine nucleosides are rather poor and much worse for the pyrimidine nucleosides. Although good yields of nucleotides are obtained by heating dry mixtures of nucleosides, ammonium oxalate or urea, and naturally occurring phosphate minerals, the availability of activated phosphates for prebiotic phosphorylation reactions, however, has been questioned based on the insolubility and scarcity of most phosphate minerals and the lability of phosphate esters. Nevertheless, the accumulated evidence suggests this is a difficult problem. Enantiomeric cross-inhibition of nucleotide stereoisomers in template polymerizations of activated RNA monomers is another hurdle for prebiotic RNA synthesis. It is worth noting that proposed RNA precursors which are achiral would not suffer from this problem, but this would postpone the problem of the introduction of
chirality into biological systems until later, perhaps once catalysts capable of discriminating between enantiomers had evolved.

Overall, it appears that the problems with the prebiotic synthesis of RNA cannot be overestimated, and thus the presence of RNA in biological systems is the result of a protracted period of abiological and biological evolution.

Our comment: Is that not remarkable? The authors conclude precisely what the evidence denies: namely that natural processes could produce RNA. 

RNA is a specialized solution to a complex set of interlocking biochemical problems: for example, the fidelity of base-pairing, the ability of the monomers to be synthesized by available catalysts and from available metabolites, the ability of large nucleic acid molecules to be folded to fit into cells, and the kinetic and thermodynamic stability of the monomers and polymers, among others. What in this view then makes RNA unique was its ability to solve multiple functional problems as they arose evolutionarily.

Our comment: Is that not a remarkable assertion ? Do prebiotic molecules have the ability of " problem solving " ? Is that not something that only a mental intelligent process can do ? Secondly, how , on what basis, can the authors conclude that evolution did the feat ? That seems to be a baseless ad-hoc assertion. The evidence actually points to the opposit direction. There was no evolution at that stage, in the first place. And secondly, natural unguided prebiotic processes would most probably not be able to produce RNA. 

These could be problems which could have been solved by other polymers but were not due to the contingencies of stochastic evolutionary processes. Alternatively, RNA may represent the best solution to these multiple selection pressures.

Our comment: Once more, the authors try to smuggle evolution into a scenario where it does not belong. There were no selective pressures to produce RNA. Why would prebiotic chemical compounds have this urge or drive ? 

As noted above, the difficulty of prebiotic RNA synthesis suggests that RNA may not have been the first informational polymer, and RNA may have been preceded by a simpler molecule. This idea is supported by studies conducted with peptide nucleic acid (PNA), which has been shown to be able to template the polymerization RNA, and glycol nucleic acid (GNA), which has also been shown to base-pair with RNA , among many other analogue structures.
The prebiotic plausibility of a primordial genetic polymer depends on several considerations, such as its ease and robustness of synthesis, stability, and functional adaptability. The relative plausibility of two or more alternative nucleic acid structures should be decided based on how these parameters compare for each molecule (Figure below ).

Proposal of primordial nucleic acid emergence

This set of criteria is by no means exhaustive. For example, first, the conditions of synthesis must be geochemically plausible, that is to say that there must be an easily demonstrable and environmentally plausible pathway for the synthesis of the monomers from small reactive intermediates such as HCN and HCHO, among others. Although they have not been investigated as extensively as RNA with regard to higher-order chemistry such as evolvable and selectable catalytic activity, peptide nucleic acid (PNA) monomers may be more plausible from the standpoint of prebiotic chemistry . Unfortunately, as mentioned above, as the conditions on the primitive Earth remain controversial, what constitutes a geochemically plausible synthesis is debatable. One possible touchstone for prebiotic chemistry comes from carbonaceous chondrites which contain a host of organic compounds formed in the primitive solar system. When compounds generated in prebiotic simulations are also found in these natural samples, their prebiotic plausibility is reinforced, although it is reasonable to suspect there were environments on the early Earth which provided conditions unlike those found in carbonaceous chondrites. Organic molecules within a compound class tend to decrease in abundance with increasing carbon number in the Murchison meteorite and in prebiotic simulations.

Thus, smaller compounds with fewer carbon atoms are likely to be more abundant, reflecting their synthesis from small precursors. For example, none of the nitrogenous bases found in RNA contain a continuous carbon chain longer than 3, although ribose contains a 5 carbon chain. 

The monomers would need some mechanism of being oligomerized. All biological nucleic acids are biosynthesized from activated precursors, nucleoside triphosphates, which overcomes the thermodynamic instability of the polymer.  DNA is considerably more stable than RNA under physiological conditions. 

In order to form a linear polymer with the ability to interact with a complementary strand, a monomer with three functionalities is required: two reactive end groups ( X and Y ) which can be linked together, and an interacting motif, B , all linked to a central backbone scaffold, b .

The minimal structural requirements for a nucleic acid monomer.

Explanations have been offered as to why phosphates (e.g., ionizability, resistance to leakage across a cell membrane, strand repulsion) and 3', 5' -linkages (e.g.,flexibility, stability) are used in biological nucleic acids and why monomers may need to be structured so as to avoid cyclization.

Base substitutions 
Early nucleic acids could  have used different bases. A variety of purine and pyrimidine derivatives have been found in prebiotic experiments. However, some of these may not be compatible with inclusion in a polymer, as some would lose their aromaticity when attached to a backbone. Some of these are rather poor at base-pairing

Alternative bases that could have been involved in the original informational polymer.

Considerable research has been conducted in this area. Many scientists have addressed base-pairing from a theoretical perspective. Earlier structures could have included alternative pyrimidines or purines, or other heterocycles that might have been more easily synthesized, more stable, or more prone to engage in self-organizational  chemistry. Bases capable of forming more than three hydrogen bonds could have been used, although the advantage of these would depend on their availability as well as the interactions these might have with the backbone and the environmental conditions such as ionic strength, pH, and temperature, all of which influence the strength and fidelity of Wattson-Crick base-pairing. For example, the GC base pair, with three hydrogen bonds, is considerably stronger than the AU or AT base pairs, with two hydrogen bonds each. Nucleic acid polymers based on different points of attachment rather than the N9 position of the purines or the N1 position of the pyrimidines have been proposed.

Watson-Crick (WC) base-pairing could be supported by other attachment points of the bases to the backbone than the normal N 9 -purine and N 1 -pyrimidine linkages.

The constant interstrand distance in DNA is maintained by pairing pyrimidines with purines. The constant interstrand distance in DNA is maintained by pairing pyrimidines with purines (Figure below ).

Various complementary ring systems could maintain constant interstrand distance. The outermost pentagonal shape represents a backbone moiety; the inner polygons represent various ring systems which allow for constant interstrand distance.

This may have been an important early selection criterion and may have limited the degree of “play” available for the testing of alternative base-pairing molecules.

Our comment: Applying a criterion of selection, and the exercise of testing, is based on goal-oriented intention, which cannot be attributed to lifeless molecules.  

Many aromatic compounds stack well in a nucleic acid context, often better than the nucleobases themselves. However, these would not be able to hydrogen bond, and it is not obvious how they would react prebiotically to attach to a linker molecule. 

Formaldehyde (systematic name methanal) is a naturally occurring organic compound with the formula CH2O (H-CHO). It is the simplest of the aldehydes (R-CHO). The common name of this substance comes from its similarity and relation to formic acid.  1

b Hydrogen cyanide (HCN), sometimes called prussic acid, is a chemical compound with the chemical formula HCN. It is a colorless, extremely poisonous and flammable liquid that boils slightly above room temperature, at 25.6 °C  2

3. Genesis - In The Beginning Precursors of Life, Chemical Models and Early Biological Evolution, page 8

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14 Abiogenesis ? No way. There is NO way, that on Fri Aug 03, 2018 10:02 pm


Abiogenesis ? No way 

There is NO way, that

- chance
- luck
- randomness
- unguided events
- non-intelligence
- natural mechanisms
- physical necessity
- chemical evolution
- self-assembly
- non external direction
- time
- Lady fortuna
- Batman
- Scooby doo
- Superman
- The Flying Spaghetti monster

could produce life by natural biochemical processes without guiding intelligence. It could not even produce ONE of the thousands of building blocks needed. As an example, lets take Thymine, one of the four bases of DNA. Why?

Thymine belongs to pyrimidine nucleotides, one of the four bases that make DNA.  The pathway to make Thymine begins with the synthesis of the base. The first reaction is performed by the Synthesis of Carbamoyl Phosphate. The substrate to begin the reaction is bicarbonate HCO3, and nitrogen donated by glutamate, and aspartate. Aspartate synthesis is a drain on the citric acid cycle Ammonia is incorporated into biomolecules through Glutamate and Glutamine. Reduced nitrogen in the form of NH4+ is assimilated into amino acids and then into other nitrogen-containing biomolecules. Two amino acids, glutamate, and glutamine, provide the critical entry point. Nitrogen fixation to obtain nitrogen used in biomolecules is an extremely complex process, depending on Nitrogenase enzymes:

The make of Nitrogenase enzymes, essential for life on earth, is a monstrously complex process

The process depends on the uptake and assimilation of Iron, Sulfur, Molybdenum, the assembly of 3 co-factors of Nitrogenase, transport systems, and so on.

Enzymes used to make Uridine Monophosphate UMP:

Carbamoyl phosphate synthase II
Aspartate carbamoyltransferase
Dihydro Orotate Dehydrogenase
Orotate Phosphoribosyl transferase
Orotidine 5'-phosphate decarboxylase
Nucleoside-phosphate kinase 
Nucleoside-diphosphate kinase

Once Uridine Monophosphate is synthesized, the further steps are

ribonucleotide reductase
nucleoside diphosphate kinase
dUTP phosphatase ( dUTPase)
thymidylate synthase
thymidylate triphosphate (dTTP)

thymidylate synthase requires:

serine hydroxymethyltransferase (SHMT)
thymidylate synthase (TS)
dihydrofolate reductase (DHFR)

and as cofactor:

N5, N10-methylenetetrahydrofolate

Tetrahydrofolate (H4 folate)has fundamental importance for the biosynthesis of purines, pyrimidines, and several amino acids.

How Do Organisms Synthesize Amino Acids?

The synthesis of this cofactor, N5, N10-methylenetetrahydrofolate, requires:

vitamin folic acid (folate)

The two essential precursors of folate biosynthesis are 4-aminobenzoate (a product of shikimate biosynthesis pathway) and GTP

The Shikimate pathway depends on following enzymes:

DAHP synthase
3-dehydroquinate synthase
5-Dehydroquinate Synthetase
Shikimate dehydrogenase
Shikimate kinase
3-enolpyruvylshikimate-5-phosphate synthase
Chorismate synthase

and GTP is Guanosine-5'-triphosphate (GTP), a purine nucleoside triphosphate. It is one of the building blocks needed for the synthesis of RNA during the transcription process.

The proteins used in the folate biosynthesis pathway are:

GTP cyclohydrolase I (GTPCH)
Dihydroneopterin aldolase (DHNA)
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase  (HPPK)
Dihydropteroate synthase (DHPS)
folylpolyglutamate synthetase (FPGS)
Dihydrofolate reductase (DHFR)

The conversion of serine to glycine is a prominent means of generating one-carbon derivatives of THF

Biosynthesis of Serine requires following enzymes:

Phosphoglycerate dehydrogenase
Phosphoserine transaminase
phosphoserine phosphatase

Not considering the enzymes to make Nitrogenase for nitrogen fixation and assimilation, almost 30 enzymes are required to make thymidylate triphosphate (dTTP) , one of the nucleotides used in DNA. 

And all these enzymes had to emerge prior life could begin. We have described the pathway to make just ONE of the four nucleobases. If someone wants to propose natural unguided processes to make all these molecular machines, the regulation, right interconnection, encoding the information to make each of these hypercomplex enzymes, the odds would be astronomically big. This is a good illustration to demonstrate, why abiogenesis is impossible. 

Design and non-design are mutually exclusive (it was one or the other) so we can use eliminative logic: if non-design is highly improbable, then design is highly probable. Thus, evidence against non-design (against production of a feature by undirected natural process) is evidence for design. And vice versa. The evaluative status of non-design (and thus design) can be decreased or increased by observable empirical evidence, so a theory of design is empirically responsive and is testable.

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15 Re: The DNA double helix - evidence of design on Tue Sep 11, 2018 12:52 pm


There is a unbridgeable gap between origin of life proposals, and the complexity of the biochemical processes in the cell to synthesize the building blocks for life.

The amazing complexity to make DNA nucleobases

Both nucleobases, Pyrimidines, and Purines had to begin to be produced prior when life began since they make up DNA - the molecule that stores genetic information. That means as well, that all enzymes used in the pathway to make the bases had to be present prior the supposed Last Universal Common Ancestor ( that's a fairy tale anyway, but for the argument, it doesn't matter )  For pyrimidines, six synthesis manufacturing/biosynthesis steps are required, and for purines, eleven. 

The thrilling part is that just one of all these enzymes is staggeringly complex. David Goodsell writes: Aspartate carbamoyltransferase is fully as complex as any fine automobile in our familiar world. 

Take just a moment to ponder the immensity of this enzyme. The entire complex is composed of over 40,000 atoms, each of which plays a vital role. The handful of atoms that actually perform the chemical reaction are the central players. But they are not the only important atoms within the enzyme--every atom plays a supporting part. The atoms lining the surfaces between subunits are chosen to complement one another exactly, to orchestrate the shifting regulatory motions. The atoms covering the surface are carefully picked to interact optimally with water, ensuring that the enzyme doesn't form a pasty aggregate, but remains an individual, floating factory. And the thousands of interior atoms are chosen to fit like a jigsaw puzzle, interlocking into a sturdy framework. Aspartate carbamoyltransferase is fully as complex as any fine automobile in our familiar world. And, just as manufacturers invest a great deal of research and time into the design of an automobile, enzymes like aspartate carbamoyltransferase are finely tuned.

Beside this enzyme, all others, almost 20, had to be produced prebiotically, and then interconnected like in a factory assembly line, to make DNA nucleobases !! 

There was no evolution. No natural selection. No mutations - nah nah  Charly won't provide the crutches to explain the feat.....

The only alternative to these biochemical processes would be, that the basic building blocks were readily available on a prebiotic earth. Glycine for instance is a indispensable substrate for pyrimidine nucleotide synthesis, and so - DNA - in cells. It  requires at least 5 biosynthetic steps and the respective enzymes to be synthesized. In a prebiotic earth, the only alternative would have been that glycine came from comets.

Comet contains glycine, key part of recipe for life 3
May 27, 2016
An important amino acid called glycine has been detected in a comet for the first time, supporting the theory that these cosmic bodies delivered the ingredients for life on Earth, researchers said Friday.
In addition to the simple amino acid glycine, the instrument also found phosphorus. The two are key components of DNA and cell membranes. "Demonstrating that comets are reservoirs of primitive material in the Solar System, and vessels that could have transported these vital ingredients to Earth, is one of the key goals of the Rosetta mission, and we are delighted with this result."

Panspermia, not a viable explanation for the OOL

Chemistry happens, and interesting molecules form in space; so what?  It’s not going to help the believers in naturalistic origin of life.  So they found glycine, the simplest and only non-chiral amino acid.  The biologists told the astronomers to look for life’s building blocks in space, because they were having such a hard time producing them on Earth.  They would need megatons of amino acids and nucleic acid bases to rain down on the Earth for any hope of getting successful concentrations, but then the precious cargo would be subject to rapid degradation by water, oxygen, UV light, and harmful cross-reactions.  Even then, they would be mixtures of left and right handed forms, with no desire nor power to organize themselves into astronomers who could invent weird science like this.
Following the unresolved issues of nucleotide biosynthesis


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