The electron carriers NAD+ and FAD accept electrons from reduced metabolites and transfer them to other compounds. Oxidation–reduction reactions (also known as redox reactions) supply living things with most of their free energy. One of the most widely occurring electron carriers are the nucleotide coenzymes nicotinamide adenine dinucleotide (NAD+)
Coenzymes and Proteins Serve as Universal Electron Carriers
The multitude of enzymes that catalyze cellular oxidations channel electrons from their hundreds of different substrates into just a few types of universal electron carriers. The reduction of these carriers in catabolic processes results in the conservation of free energy released by substrate oxidation. NAD, NADP, FMN, and FAD are water-soluble coenzymes that undergo reversible oxidation and reduction in many of the electron- transfer reactions of metabolism. The nucleotides NAD and NADP move readily from one enzyme to another; the flavin nucleotides FMN and FAD are usually very tightly bound to the enzymes, called flavoproteins, for which they serve as prosthetic groups. 7
NAD is pivotal for cell life, first as a reusable redox coenzyme for energy production, second as a consumable substrate in enzymatic reactions regulating crucial biological processes, including gene expression, DNA repair, cell death and lifespan, calcium signaling, glucose homeostasis, and circadian rhythms 5 Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components. NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. Most importantly, it is also the driving force of most biosynthetic enzymatic reactions, including those responsible for the biosynthesis of all major cell components, such as DNA and lipids. Traditionally, the dehydrogenase reactions of the oxidative pentose phosphate pathway (oxPPP), the Entner–Doudoroff (ED) pathway, and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle have been considered the major sources of NADPH. However, the importance of other NADPH-generating enzymes, such as transhydrogenases, glucose dehydrogenases, and non-phosphorylating glyceraldehyde 3- phosphate dehydrogenase (GAPN), is becoming clear, indicating that the traditional view is over-simplistic. The consumption of NADP+ is connected to the consumption of NAD+ and to the regulation of various major biological activities such as DNA repair, gene expression, apoptosis, nitrogen fixation, and calcium homeostasis. 6 Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components 8
NADH and NADPH Act with Dehydrogenases as Soluble Electron Carriers
Nicotinamide adenine dinucleotide (NAD; NAD+ in its oxidized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP; NADP when oxidized) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond. The vitamin niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. Both coenzymes undergo reversible reduction of the nicotinamide ring. As a substrate molecule undergoes oxidation (dehydrogenation), giving up two hydrogen atoms, the oxidized form of the nucleotide (NAD+ or NADP+) accepts a hydride ion (:H, the equivalent of a proton and two electrons) and is reduced (to NADH or NADPH). The second proton removed from the substrate is released to the aqueous solvent. In the abbreviations NADH and NADPH, the “H” denotes the added hydride ion. To refer to these nucleotides
without specifying their oxidation state, we use NAD and NADP. NAD+ generally functions in oxidations—usually as part of a catabolic reaction; NADPH is the usual coenzyme in reductions—nearly always as part of an anabolic reaction. More than 200 enzymes are known to catalyze reactions in which NAD+ (or NADP+) accepts a hydride ion from a reduced substrate, or NADPH (or NADH) donates a hydride ion to an oxidized substrate. 7
Two of the most widely occurring electron carriers are the nucleotide coenzymes nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD). The nicotinamide portion of NAD+ (and its phosphorylated counterpart NADP+; Figure below)
The Nicotinamide Coenzymes
NAD1/NADH and NADP1/NADPH carry out hydride transfer reactions. All reactions involving these coenzymes are two-electron transfers 3 The structures and reaction of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). Their reduced forms are NADH and NADPH. These substances, which are collectively referred to as the nicotinamide coenzymes or pyridine nucleotides (nicotinamide is a pyridine derivative), function as intracellular electron carriers. Reduction (addition of electrons) formally involves the transfer of two hydrogen atoms (H·), or a hydride ion and a proton (H:− + H+). Note that only the nicotinamide ring is changed in the reaction.
It is the site of reversible reduction, which normally occurs as the transfer of a hydride ion (H−; a proton with two electrons) as indicated in Figure below
Reduction of NAD+ to NADH. R represents the ribose–pyrophosphoryl–adenosine portion of the coenzyme.
Only the nicotinamide ring is affected by reduction, which is formally represented here as occurring by hydride transfer
The terminal electron acceptor in aerobic organisms, O2, can accept only unpaired electrons (because each of its two available lowest energy molecular orbitals is already occupied by one electron); that is, electrons must be transferred to O2 one at a time. Electrons that are removed from metabolites as pairs (e.g., with the two-electron reduction of NAD+) must be transferred to other carriers that can undergo both two-electron and one-electron redox reactions. FAD is such a coenzyme.
The reactive part of NAD+ is its nicotinamide ring, a pyridine derivative synthesized from the vitamin niacin. In the oxidation of a substrate, the nicotinamide ring of NAD+ accepts a hydrogen ion and two electrons, which are equivalent to a hydride ion (H: - ) The reduced form of this carrier is called NADH . In the oxidized form, the nitrogen atom carries a positive charge, as indicated by NAD+ . NAD+ is the electron acceptor in many reactions of the type
Coenzymes likely represent the oldest metabolic fossils within a cell, as suggested by their presence and essentiality in all kingdoms of life and the autocatalytic nature of their biosynthetic pathways. The presence of a ribonucleotidyl group in the structure of most coenzymes that use it as a “handle” for binding to the protein catalyst means that ribonucleotides must have been present at the time coenzymes emerged. An open question remains whether the ribonucleotidyl group has been co-opted from a preexisting RNA in a primordial “RNA world” before the emergence of proteins, or it represents the evolutionary predecessor of contemporary nucleic acids. The nicotinamide coenzyme NAD (P) is one of the oldest molecules, not only in the history of biochemistry, but also in the evolutionary steps towards the emergence of life. Together with relatively simple organics, such as PRPP (50-phosphoribosyl 10-pyrophosphate), PLP (pyridoxal 50-phosphate) and many others, it may have been a crucial prebiotic agent in organizing a collectively autocatalytic protometabolic ecosystem. Here, the NAD(P) biosynthetic pathway will be described with views on its origin. NAD(P)’s peculiar biochemical features will also be discussed with the aim to offer novel arguments to the debate on the sequence of chemical evolution in the origin of life. 1
Coenzymes are ubiquitous and small molecules required by many protein enzymes to catalyze reactions that are otherwise inefficient or impossible for typical amino acids. By binding to the catalyst, either through covalent or weak interactions, they function as transient carriers of specific functional groups. In cellular metabolism, the same coenzyme is usually used by enzymes with different substrate specifities; for example, NAD(P) is used as electron carrier by several cellular dehydrogenases a catalyzing reduction and oxidation of a wide variety of substrates. Given their essential function and broad use, it is evident that a change in coenzyme structure would inevitably result in a fatal outcome for the organism. Therefore, it is reasonable to assume that, in evolutionary time, once coenzymes had gained the most suitable structure for their function, they should have retained it more stringently than the catalysts. As a consequence, contemporary coenzymes should not be much different from their ancestral precursors. Indeed, they are considered the oldest metabolic fossils within a cell. The autocatalytic nature of coenzymes also speaks in favor of an ancient metabolic history. An autocatalytic metabolite is a compound which is required for its own synthesis and cannot be accessed from the food set.
Well known is the autocatalytic nature of ATP: in glycolysis, the nucleotide is essential to kick-start the pathway, but once glycolysis has been launched, ATP is able to support the pathway and thus its own synthesis. A recent research based on the metabolic reconstruction of coenzyme biosynthetic pathways in various eubacterial organisms has provided evidence that also some coenzymes, including NAD, coenzyme A and tetrahydrofolate, behave as autocatalytic molecules, that is they are able to support their own synthesis.
Nicotinamide adenine dinucleotide (NAD) is a pivotal cofactor in the cell. It consists of nicotinamide mononucleotide (NMN) and adenosine monophosphate, linked by a pyrophosphate bond. 4
Notably, some coenzymes, including NAD, FAD, coenzyme A and adenosylcobalamin, share a ribonucleotidyl group, even though they have completely different biochemical roles. Indeed, in none of them does the ribonucleotidyl moiety participate directly in the coenzymatic function; it rather acts as a “handle” for binding to the protein catalyst, as revealed by a structural analysis of enzymes in complex with ATP, NAD(P), FAD and coenzyme A . The analysis shows that different proteins recognize the adenine moiety of coenzymes with a common binding motif. Notably, binding to adenine relies on polar and hydrophobic interactions, which are not dependent on the protein sequence and very closely resemble adenine base-pairing and -stacking in DNA and RNA. The presence of such a motif in ancient proteins, which are present in all living organisms, suggests that it has been exploited very early in biotic evolution.
The occurrence of a ribonucleotidyl moiety in cofactors means that ribonucleotides must have been present at the time coenzymes, as we know them, emerged. A question is whether the ribonucleotidyl group has been co-opted from an existing RNA or it represents the evolutionary predecessor of contemporary nucleic acids. The first assumption presupposes the existence of a primordial “RNA world”, which is believed to be a time before the emergence of template-encoded proteins when metabolism was guided entirely by RNA. In this scenario, nucleotidyl coenzymes might have been synthesized by RNA enzymes and used by them to support all the different types of reactions that are not feasible with only the four nucleotides. The biochemical properties of RNA are indeed compatible with this scenario, as revealed by the in vitro selection through directed evolution of RNAs able to synthesize ribonucleotide coenzymes and RNAs able to bind coenzymes and use them to perform the chemical reactions typical of modern protein enzymes. Moreover, the occurrence of natural small eubacterial RNAs carrying at their 50 termini coenzymes like NAD and coenzyme A (Chen et al. 2009; Kowtoniuk et al. 2009), together with the presence in all living organisms of riboswitches that directly bind to, and hence are regulated by thiamine pyrophosphate, flavin mononucleotide (FMN) and adenosylcobalamin bring important insights into the plausibility of the above hypothesis. Based on the consideration that precursors of some coenzymes, like NAD and coenzyme A, can be synthesized from simple molecules under prebiotic conditions, one line of reasoning is that the early chemical environment contained functionalized molecules similar to modern coenzymes, but without the nucleotidyl moiety. These molecules would have been highly reactive and initiated a series of chemical reactions. Later on, catalyst RNAs appeared which adopted them to expand their catalytic repertoire, thus initiating coenzyme-RNA-dependent metabolic pathways. In this view, modern nucleotidyl coenzymes might have originated as coribozymes and the adenine ribonucleotide moiety might represent a surviving vestige of primordial RNA.
NADPH-Generating Reactions Coupled to Central Carbon Metabolism
The oxidative pentose phosphate pathway (oxPPP) enzymes Glucose-6-phosphate dehydrogenase (G6PDH) and Phosphogluconate dehydrogenase ) have a central role in metabolism in many microbes. The enzymes are involved in the conversion of glucose-6-phosphate into ribulose-5-phosphate, a precursor of important molecules such as nucleic acids, and are generally considered a major source of NADPH. The importance of the oxidative pentose phosphate pathway ( oxPPP ) enzymes as a source of NADPH has been demonstrated in various organisms. The key enzyme with regard to the control of PPP flux is thought to be G6PDH.
A peculiar feature of NAD biosynthesis is that in extant living species different combinations of the metabolic routes are operative, depending on the organism. This results in a great diversity of the NAD biosynthetic machinery in various species, implying some evolutionary complexity. 6
Had this machinery and the central metabolic pathways not to emerge prior when life began, since life depends on NADPH , and as such, puts common ancestry into question?
Nicotinamide Coenzyme Biosynthesis
Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. 8
In contemporary metabolism, NAD(P) can be synthesized both de novo, starting from simple precursors, and through salvage routes that allow NAD synthesis from both the nicotinamide liberated by the NAD-consuming enzymes and the pyridine ring that is available from the food set, in the form of nicotinamide and nicotinic acid (also known as vitamin B3). Two alternative de novo routes exist, one starting from aspartate and dihydroxyacetone phosphate, the other from tryptophan.
The former is operative in most bacteria, archaea and plants; the latter occurs in animals, including humans, fungi and few bacterial species. Both routes converge to the formation of quinolinate. NAD synthesis from quinolinate is depicted in Fig. 9.2. Quinolinate is first converted to nicotinate mononucleotide (NaMN) by the enzyme quinolinate phosphoribosyltransferase that uses 50-phosphoribosyl 10-pyrophosphate (PRPP) as the phosphoribosyl (PR) donor. NaMN is then converted to NAD by NaMN adenylyltransferase and NAD synthetase that catalyze two consecutive reactions, common to both salvage and de novo pathways. ATP-dependent phosphorylation of NAD by NAD kinase leads to NADP formation. Figure 9.2 also shows the most common salvage routes: nicotinamide can be deamidated to nicotinic acid via nicotinamidase, followed by the PRPPdependent phosphoribosylation of nicotinic acid to NaMN by nicotinate phosphoribosyltransferase; alternatively, nicotinamide can be directly converted to NAD via two consecutive reactions catalyzed by nicotinamide phosphoribosyltransferase that uses PRPP to form nicotinamide mononucleotide (NMN), and NMN adenylyltransferase which adenylates NMN to NAD. A salvage pathway also occurs in which NAD is synthesized from the pyridine nucleosides nicotinamide riboside and nicotinate riboside, after their conversion to the corresponding mononucleotides (not shown). 1
NADPH generating enzymes coupled to central carbon metabolism
Glucose-6-phosphate dehydrogenase (G6PD or G6PDH)
PGD - phosphogluconate dehydrogenase
Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)
Glyceraldehyde-3-phosphate dehydrogenase (NADP+)
Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating)
Two principal NAD+ biosynthesis pathways have been characterized: (1) the de novo pathway and (2) the salvage pathway. In the de novo pathway, NAD+ is generated from quinolinic acid, which in prokaryotes is produced from either L-aspartate or L-tryptophan. In the salvage pathway, degradation products containing a pyridine ring, namely nicotinic acid and nicotinamide, are utilized to regenerate NAD+. ( Figure A, below )
NAD+ and NADP+ biosynthetic pathways in prokaryotes.
(A) De novo and salvage pathways for NAD+ biosynthesis.
(B) NADP+ biosynthesis by NAD kinase.
NaMN, nicotinic acid mononucleotide;
NaAD, nicotinic acid adenine dinucleotide;
NR, nicotinamide riboside;
NMN, nicotinamide mononucleotide;
NAD, nicotinamide adenine dinucleotide;
NADP, nicotinamide adenine dinucleotide phosphate;
NADK, NAD kinase.
Schematic overview of major NAD biosynthetic routes starting from the free pyridine ring.
The pyridine ring is used in the form of quinolinate (Qa), nicotinate (Na), nicotinamide (Nam). Enzymes catalyzing the reactions are:
1. Quinolinate phosphorybosyltransferase
2. NMN/ NaMN adenylyltransferase
3. NAD synthetase
4. Nicotinate phosphoribosyltransferase
5. nicotinamide deamidase
6. nicotinamide phosphoribosyltransferase
Other abbreviations used are: DHP diydroxyacetone phosphate, NaMN nicotinate mononucleotide), NaAD
nicotinate adenine dinucleotide, NMN nicotinamide mononucleotide, PRPP 50-phosphoribosyl
Phylogenetic and structural studies involved in NAD biosynthesis suggest that quinolinate phosphoribosyltransferase (enzyme 1 in Figure above) is supposedly the most ancient: it might have given rise to nicotinate phosphoribosyltransferase (enzyme 4 in Figure above), which in turn supposedly evolved to nicotinamide phosphoribosyltransferase (enzyme 6 in Figure above). It is therefore likely that primordial cells synthesized NAD de novo starting from aspartate and only later the salvage routes emerged. A phylogenetic analysis of the two enzymes salvaging nicotinamide, that is nicotinamide deamidase (enzyme 5 in Figure above) and nicotinamide phosphoribosyltransferase, points to an earlier emergence of the former. The gene coding for the deamidase is, in fact, present in a larger variety of living species and is distributed more deeply in the tree of life while the gene coding for the PR transferase is present only in few eubacterial species, bacteriophages, sponges and vertebrates. Accordingly, NAD biosynthesis from nicotinamide through the deamidated pathway might have predated the coenzyme synthesis through the amidated one. Notably, among the few organisms using nicotinamide phosphoribosyltransferase are some a-proteobacteria which are considered relatives of the first mitochondria. Indeed, in vertebrates a mitochondrial homolog of the enzyme is present, which is essential for maintaining physiological levels of mitochondrial NAD under genotoxic stress and nutrient restriction. Under these conditions, in fact, mitochondrial nicotinamide phosphoribosyltransferase is upregulated and the increased enzymatic activity provides protection against cell death. This suggests that NAD levels might have controlled cell survival in the bacteria that gave rise to mitochondria, and the survival pathway might have been conserved up to the present day in vertebrates.
A portion of the synthesized NAD+ can be converted into NADP+ by NAD kinase (NADK) (Figure B above). In the NAD+ synthesis pathways, several variations exist and multiple enzymes are involved. In contrast, NADK is the sole enzyme able to generate NADP+ de novo. NAD kinase is found in archaea, bacteria, and eukaryotes and has been proven to be essential in prokaryotes. According to the literature, only one species not able to synthesize NADP+ from NAD+ has been identified: Chlamydia trachomatis. The intracellular parasite appears to lack NADK and hence relies completely on the metabolism of its host cell. NADK has received much attention since its discovery in 1950.
Pyridine Ring Prebiotic Synthesis
Synthesis of the coenzyme in prebiotically plausible conditions still remains a matter of speculation. Both nicotinamide and nicotinic acid can be successfully produced from inorganic elements in experiments simulating early-Earth conditions. In particular, nicotinonitrile, which hydrolyzes to nicotinamide and nicotinic acid, can be synthesized under the action of an electric discharge on ethylene and ammonia or from the reaction of cyanoacetaldehyde, propiolaldehyde and ammonia, which are in turn synthesized from a spark discharge. An alternative scenario for a prebiotic synthesis of the pyridine ring has been suggested following the discovery of an easy and efficient way to nonenzymatically generate the pyridine ring in the form of quinolinate starting from the amino acid aspartate and ribose or deoxyribose.
Of course it would have to be asked then where the aspartate or ribose or deoxyribose came from on a prebiotic earth.
Studies of the intermediates of the reactions involved in this process suggest that methylglyoxal, which is one of the major acid degradation products of both ribose and deoxyribose, reacts with aspartate in a five-step nonenzymatic sequence of reactions, yielding quinolinate and, to a lesser extent, nicotinate.
It can reasonably be assumed that the extant metabolic pathway resulted from a preexisting pathway, with gradual enzymatic takeover of the various steps. The pyridine ring might have arisen independently of enzymatic
chemistry and provide evidence for the presence and involvement of the pyridine coenzymes in the earliest metabolic system.
This is a typical just-so ad-hoc assertion based on blind faith and guesswork. No evidence exists to back up the claim whatsoever. There is a huge gap between the proposed abiotic, non - enzymatic scenarios, and the modern metabolic pathways by several complex enzymes, which had to emerge by pure luck and chance since evolution was not extant at this stage.
A Direct Prebiotic Synthesis of Nicotinamide Nucleotide
The “RNA World” hypothesis proposes an early episode of the natural history of Earth, where RNA was used as the only genetically encoded molecule to catalyze steps in its metabolism.
This, according to the hypothesis, included RNA catalysts that used RNA cofactors.
However, the RNA World hypothesis places special demands on prebiotic chemistry, which must now deliver not only four ribonucleosides, but also must deliver the “functional” portion of these RNA cofactors.
While some (e.g., methionine) present no particular challenges, nicotinamide ribose is special.
Essential to its role in biological oxidations and reductions, its glycosidic bond that holds a positively charged heterocycle is especially unstable with respect to cleavage.
Nevertheless, we are able to report here a prebiotic synthesis of phosphorylated nicotinamide ribose under conditions that also conveniently lead to the adenosine phosphate components of this and other RNA cofactors. 9
a A dehydrogenase (also called DH or DHase in the literature) is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. They also catalyze the reverse reaction, for instance alcohol dehydrogenase not only oxidizes ethanol to acetaldehyde in animals but also produces ethanol from acetaldehyde in yeast. 2
1. Origins of Life: The Primal Self-Organization
3. Biochemistry 6th ed. Garrett, page 647
7. Lehninger, Principles of biochemistry, 6th ed. page 516