The Rubisco enzymes amazing evidence of design

Rubisco's amazing evidence of design  

https://reasonandscience.catsboard.com/t1554-the-rubisco-enzymes-amazing-evidence-of-design

Although RubisCO has been intensively studied, its evolutionary origins and rise as Nature’s most dominant carbon dioxide (CO2)-fixing enzyme still remain in the dark. 4

Rubisco is the most important enzyme on the planet. virtually all the organic carbon in the biosphere derives ultimately from the carbon dioxide that this enzyme fixes from the atmosphere. Without it, advanced life would not be possible. And we would not be able to debate our origins. All inquiry and quest about if we are ultimately the result of a powerful creator, or just random natural chemical reactions and emerging properties of lifeless matter, if biodiversity is due to evolution, or an intelligent designer, is second to the inquiry of how Rubisco came to be.  Rubisco has a  complex structure, functioning and synthesis process, many cell compartments, enzymes, proteins, and pathways are involved and required to assemble it,  the unfinished sub-units require co and post-translational modifications, specific proteins that help like assembly robots in the manufacturing process, sophisticated pathways, and mechanisms of protein import and targeting in chloroplasts through large multi-protein translocon complexes in the stroma, and advanced protein communication and information systems. All this is of bewildering complexity, where dozens of individual interconnected and finely tuned parts are required, a web of interlocked extremely complex advanced molecular machines where if one is missing, nothing goes, that defy the intelligence of the best scientists for decades to find out their structure, mechanisms, and functions. Could all this be due to natural processes?

RuBisCO is a multi-subunit plant protein essential to photosynthesis.  It catalyzes the primary chemical reaction by which inorganic carbon enters the biosphere. In the C3 pathway, RuBisCO is responsible for initiating the first step in carbon dioxide fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. This step of the Calvin Cycle plays a crucial role in providing energy for the cell.

https://reasonandscience.catsboard.com/t2164-the-calvin-benson-cycle

Rubisco is also the most abundant enzyme on earth. It is present in every plant and photosynthetic organism, from the smallest cyanobacteria and plankton to palm trees and giant sequoias. Rubisco is a complex composed of eight large subunits and eight small subunits

Synthesized RuBisCO does not have a fully functional active site. It needs to be activated by a CO2 molecule that carbamylates its catalytic Lys to bind Mg2+ that completes the activation process... The carboxylation involves at least four, perhaps five discrete steps and at least three transition states;

The origin of these highly specific, regulated, and coordinated steps, which are essential for the activation of Rubisco, is best explained through a planning mind, which all set it up. Natural mechanisms are extremely unlikely to be capable to produce these sophisticated metabolic multistep pathways and assembly lines to make Rubisco in the first place. No wonder, that no mainstream scientific papers are able to provide compelling evolutionary scenarios.    As long as the enzyme is not fully functional, nothing goes, and ultimately, advanced life on earth would not be possible. How did the correct insertion of the correct metal cation Mg2+ surrounded by three H2O/OH molecules emerge? Trial and error? The genome needs the right information in order to get the right materials, the right shape and quantity of each subunit co-factors and metal clusters, how to position them at the right active site, and how to mount these parts in the right order. That seems to me only being explained in a compelling manner by the wise planning of a super-intelligent engineer, which knew how to invent and build this highly sophisticated and complex machine and make it fully functional right from scratch. A stepwise unguided emergence seems to be extremely unlikely. This mechanism seems to be the result of intelligence, which sets it all up through power, will, and information.

The eight large subunits of Rubisco are coded by the chloroplast DNA and the eight small subunits by nuclear DNA. The small subunit of Rubisco and all the other Calvin cycle enzymes are encoded by nuclear genes and must be transported and travel to the chloroplast site after their synthesis in the cytosol.

https://reasonandscience.catsboard.com/t2165-pathways-and-mechanisms-of-protein-import-and-targeting-in-chloroplasts

The precursor forms of these stromal proteins contain an N-terminal stromal-import sequence. This transit peptide allows transfer of the small subunits synthesized in the cytosol through the chloroplast envelope translocon complexes into the plastid. These are highly complex molecular gates in the chloroplast inner and outer membrane, which filter which molecules go in.  After the unfolded precursor enters the stromal space, it binds transiently to a stromal Hsc70 chaperone and the N-terminal sequence is cleaved.

Folding of the small and large Rubisco subunit proteins is mediated by the amazing GroEL–GroES chaperonin system. Protein folding mediated by chaperonins is the process by which newly synthesized polypeptide chains acquire the three-dimensional structures necessary for biological function. For many years, protein folding was believed to occur spontaneously.  But it has become apparent that large proteins frequently fail to reach native state, forming nonfunctional aggregates instead. They need the aid of these sophisticated barrel-shaped proteins.

https://reasonandscience.catsboard.com/t1437-chaperones?highlight=chaperones

That raises interesting questions: How should and could natural non-guided natural mechanisms foresee the necessity of chaperones in order to get a specific goal, that is the right precise 3-dimensional folding resulting in functional proteins to make living organisms? The nonliving matter has no natural " drive " or purpose or goal to become living. The make of proteins to create life, however, is a multistep process of many parallel acting complex metabolic pathways and production-line like processes to make proteins and other life-essential products like nucleotides, amino acids, lipids, carbohydrates, etc. The right folding of proteins is just one of several other essential processes in order to get a functional protein. But a functional protein by its own has no function unless correctly embedded in the right assembly sequence and order at the right functional place.

Eight S subunits combine with the eight L subunits to yield the active rubisco enzyme. At least three chloroplast outer membrane proteins, including a receptor that binds the stromal-import sequence and a translocation channel protein, and five inner-membrane proteins are known to be essential for directing proteins to the stroma. Import into the stroma depends on ATP hydrolysis catalyzed by a stromal Hsc70 chaperone. Chloroplasts cannot generate an electrochemical gradient (proton-motive force) across their inner membrane. Thus protein import into the chloroplast stroma is powered solely by ATP hydrolysis. Within the stroma, the S-subunits undergo further posttranslational modification (transit peptide cleavage, Met-1 aN- methylation) prior to assembly into final L8S8 Rubisco complexes. How did natural evolutionary processes find out how to do it? Trial and error?

In order to make and assemble Rubisco, at least  25 parts, most of the essential and irreducible, are directly involved in Rubisco function, activation, and  synthesis:

https://reasonandscience.catsboard.com/t1554-the-rubisco-enzymes-amazing-evidence-of-design#3899

Could these parts, proteins enzymes, etc.  have evolved separately and gradually?  What about the RbcX Assembly Chaperone, specifically used as an assembly tool of Rubisco? What about the barrel-shaped GroEL/GroES chaperonins which perform their function with extremely impressive simplicity and elegance, namely helping over 100 different proteins to get into their correct shape and form, essential for function? ( in our case, helping the Rubisco RbcL subunits to get their proper shape )

These chaperone systems are themselves made of proteins which also require the assistance of chaperones to correctly fold and to maintain integrity once folded. Chaperones for chaperones in fact. The very simplest of cells that we know of have these systems in place.

Or how do proponents of evolution explain how natural selection would have favored the emergence of Hsp70 chaperones,  central components of the cellular network,  proteins which assist a large variety of protein folding processes in the cell by the transient association of their substrate-binding domain with short hydrophobic peptide segments within their substrate proteins?  That is in our case, their function of which was to prevent a still-useless rubisco small subunit from folding outside the chloroplast?  They are made, used during the synthesis process, and once Rubisco assembly has finished, these enzymes are discarded. This is very much a factory-like production and assembly-line process, using fully automatized and programmed nano-robot like molecular machines, namely enzymes.  Most parts, if missing, render 1. the assembly of Rubisco impossible, and 2. Rubisco useless. Many parts, if missing render it not fully functional and defective.  Besides the enzymes that have used in other biological systems, there would be no reason to make them unless all other parts were there too, and the assembly instructions of Rubisco. As an analogy, if you had to make the implementation of a car factory, why would you make the assembly chain of a piston, if you do not have all the precise instructions to make 1. the car as a whole, and 2. the instructions of the precise shape and the materials required for the piston in particular, and how to mount it in the motor? That's precisely what happens in the cell. Evolution has no consciousness, and no foresight nor intelligence. But precisely that is required for PLANNING and make of blueprints. I cannot create a machine, without the precise drawing and project information in advance, which is required to make 1. the assembly tools 2. the subparts 2. the whole machine.

How do proponents of evolution explain how natural selection would have favored a protein complex the function of which was to prevent a still-useless Rubisco small subunit from folding outside the chloroplast? Before it evolved a way to get the protein inside, there would be no benefit from keeping it unfolded outside. How could blind chance ‘know’ it needed to cause large subunit polypeptides to fold ‘correctly’ and to keep them from clumping? It could not ‘anticipate’ the ‘correct’ confirmation before the protein became useful. And evolution would need to be clever indeed to chemically modify something not yet useful so that it could be folded ‘correctly’ when even the ‘correctly’ folded polypeptide would not yet become useful.


Only a designer would know why it would be necessary
to produce a specialized protease, target it to the chloroplast, and program it to clip off the targeting sequence of the small subunit at just the right place. And what about the assembly of a collection of meaningless rubisco parts in just one certain way? In order to design a sophisticated set of tools to make something else useful in the future that had, as yet, no function, evolution (as ‘designer’) would have had to have detailed knowledge of the future usefulness of the protein it was so cleverly engineering. If evolution managed to generate any one of these chaperone protein complexes (and it would not), it would still be useless for generating rubisco unless all the other chaperones were also present. Without any one of them, the sixteen-unit complex could not be generated.

That totally destroys the Evolution Theory:  How should and could natural nonguided natural mechanisms foresee the necessity of chaperones in order to get a specific goal, that is the right precise 3-dimensional folding resulting in functional proteins to make living organisms? The nonliving matter has no natural " drive " or purpose or goal to become living. The make of proteins to create life, however, is a multistep process of many parallel acting complex metabolic pathways and production-line like processes to make proteins and other life-essential products like nucleotides, amino acids, lipids, carbohydrates, etc. The right folding of proteins is just one of several other essential processes in order to get a functional protein. But a functional protein on its own has no function unless correctly embedded in the right assembly sequence and order at the right functional place." that's precisely the problem of evolution. there is no foresight. So why would evolution produce an assembly chaperone enzyme to make rubisco? You don't make a robot for an assembly line if the end product is not known.


(RuBisCO) is a multi-subunit plant protein essential to photosynthesis.  It catalyzes the primary chemical reaction by which inorganic carbon enters the biosphere. In the C3 pathway, RuBisCO is responsible for initiating the first step in carbon dioxide fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. In chemical terms, it catalyzes the carboxylation of ribulose-1,5-bisphosphate (also known as RuBP).3 This  step of the Calvin Cycle plays a crucial role in providing energy for the cell. It is the most important enzyme on the planet — virtually all the organic carbon in the biosphere derives ultimately from the carbon dioxide that this enzyme fixes from the atmosphere. 3 Rubisco is also the most abundant enzyme on earth. It is present in every plant and photosynthetic organism, from the smallest cyanobacteria and plankton to palm trees and giant sequoias. Rubisco is a complex composed of eight large subunits and eight small subunits  2




RuBisCO vs alternative carbon fixation pathways

While many autotrophic bacteria and archaea fix carbon via the reductive acetyl CoA pathway, the 3-hydroxypropionate cycle, or the reverse Krebs cycle, these pathways are relatively smaller contributors to global carbon fixation than that catalyzed by RuBisCO. Phosphoenolpyruvate carboxylase, unlike RuBisCO, only temporarily fixes carbon. Reflecting its importance, RuBisCO is the most abundant protein in leaves, accounting for 50% of soluble leaf protein in C3 plants (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in C4 plants (5–9% of total leaf nitrogen).

Structure

In plants, algae, cyanobacteria, and phototrophic and chemoautotrophic proteobacteria, the enzyme usually consists of two types of protein subunit, called the large chain (L) and the small chain (S).






Structural view of the RubisCO enzyme. A) Front view, with the large subunits in blue/yellow and the small subunits in purple; B) Top view, with the central solvent channel visible; C) Front view, with the two large subunits (in ribbons) forming the catalytic dimer; D) Sites detected under functional divergence.



Conserved structural features of Rubisco.

A Spinach L8S8 Rubisco (Protein Data Bank 8RUC) drawn using Pymol to highlight arrangement of the S-subunits (blue) capping the catalytic core of four L2 subunits (green).

B Structural details for one L-subunit of an L2 pair highlighting one active site within the α/β-barrel of the C-terminal domain (green ribbons) and residues in the N-terminal domain (yellow ribbons) that contribute to the second active site in each L2.

C Arrangement of the conserved Rubisco active-site residues within a L-subunit C-terminal domain relative to carbamylated Lys-201 (K201X), bound Mg2+, and the six-carbon reaction intermediate mimic, 2-carboxyarabinitol 1,5-bisphosphate (CABP). The activating CO2 in K201X and the approximate positioning of substrate CO2 that binds to C-2 of the RuBP enediol are highlighted in ball-and-stick representations. Residues are numbered relative to spinach Rubisco. The conserved active site residues Glu-60 and Asn-123 from the N-terminal domain of the paired L-subunit are not shown.

From a structural point of view, all Rubisco enzymes comprise at least two large (L-) subunits. Despite there being as little as 30% amino acid identity between the different Rubisco forms, they all show a conserved L-subunit structure comprising an N-terminal domain (approximately 150 amino acids) and a larger C-terminal domain (approximately 320 amino acids) that forms an α/β-barrel . Paired L-subunits arrange head to tail to form a dimer (L2), with two active sites located at the L-L interface. The highly conserved catalytic residues predominantly reside within the α/β-barrel domain, with a few residues supplied by the N-terminal domain of the adjacent L-subunit.

Different organisms have diverse arrangements of the Rubisco L2 building blocks. Form I Rubiscos are the most abundant form found in plants, algae, and many photosynthetic bacteria. The L2 subunits in form I Rubiscos are arranged in an (L2)4 core, with two groups of four small (S-) subunits capping the L8 core to form an L8S8 molecule (Fig. A above). Although not strictly required for CO2 fixation, the S-subunits are essential for maximal activity and provide structural stability. Rubiscos classed as forms II and III lack S-subunits, containing only L-subunits arranged into L2 to (L2)5 complexes. These Rubiscos are primarily found in phototrophic proteobacteria, chemoautotrophs, dinoflagellates, and achaea.

Although the classification of Rubisco enzymes into forms I, II, and III is generally supported by sequence phylogenies, quaternary structures, and functional properties, there are exceptions.

The large-chain gene (rbcL) is part of the chloroplast DNA molecule in plants.
There are typically several related small-chain genes in the nucleus of plant cells, and the small chains are imported to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast membrane.

The enzymatically active RUBISCO substrate  binding sites are located in the large chains that form dimers in which amino acids from each large chain contribute to the binding sites. A total of eight large-chains (= 4 dimers) and eight small chains assemble into a larger complex.

Magnesium ions (Mg2+) are needed for enzymatic activity. Correct positioning of Mg2+ in the active site of the enzyme involves addition of an "activating" carbon dioxide molecule (CO2) to a lysine in the active site (forming a carbamate). Formation of the carbamate is favored by an alkaline pH. The pH and the concentration of magnesium ions in the fluid compartment (in plants, the stroma of the chloroplast) increases in the light.

1) http://www.eplantscience.com/index/algae/photosynthesis/light_independent_reactions.php#aa
2) https://en.wikipedia.org/wiki/RuBisCO
3) https://en.wikipedia.org/wiki/RuBisCO
4. https://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S095816691730099X

RUBISCO CATALYSIS 1

A Bifunctional Enzyme That Catalyzes Multistep Chemistry

Despite amino acid sequence variability within the Rubisco family, key active-site residues are absolutely conserved among forms I, II, and III Rubiscos . As a result, the activation process and complex catalytic chemistry are also preserved, despite the different biological roles of forms I and II Rubiscos, which initiate primary carbon assimilation, and the catabolic role of archaeal form III enzymes, which remove RuBP produced during purine/pyrimidine metabolism . Most structure-function studies have focused on Rubiscos involved in carbon assimilation and provide much of our mechanistic understanding of its catalysis.

Prior to catalysis, Rubisco needs to be preactivated via the reaction of a CO2 molecule with a conserved active-site Lys (residue 201 in most plant Rubisco L-subunits) to form a carbamate, which is then stabilized by Mg2+ binding (Fig. C). Following activation, Rubisco can productively bind RuBP and catalyze a complex five-step reaction that adds a CO2 and a water molecule to RuBP, followed by its cleavage and release of two 3-phosphoglycerate (3PGA) molecules



Simplified scheme illustrating how CO2 fixed to RuBP by Rubisco is distributed among the resulting two molecules of 3PGA that feed into the photosynthetic Calvin cycle to produce triose phosphates (glyceraldehyde 3-phosphate [G3P]) for carbohydrate synthesis or RuBP regeneration. The contrasting oxygenation reaction of Rubiscos produces 2-phosphoglycolate (2PG), which requires the photorespiratory pathway to recycle it back to 3PGA. Photorespiration is a complex pathway that involves four subcellular compartments and multiple enzymatic steps (represented by dashed lines), requires additional energy (ATP), and results in a loss of fixed CO2 in the mitochondria.

The complexity of the multistep process can lead to unwanted side reactions that result in the formation of inhibitors such as xyulose-1,5-bisphosphate . Furthermore, the electrostatic similarity between O2 and CO2 and their disproportionate atmospheric abundance (21% O2, 0.04% CO2) make it hard for Rubisco to totally discriminate between them, resulting in the unwanted oxygenation of RuBP and the production of one molecule of 3PGA and one of 2-phosphoglycolate. In plants, 2-phosphoglycolate is recycled back to 3PGA via photorespiration, an energy-consuming process (e.g. ATP) that liberates fixed carbon as CO2.

Several groups have applied up-to-date computational tools to examine the energetics and atomistic details of Rubisco’s carboxylation and oxygenation reactions. While these calculations reveal molecular details of the contribution of active-site residues to Rubisco catalysis, the large size of Rubisco prevents the inclusion of all atoms in the calculation. As a result, the design of “better” Rubiscos using in silico modeling tools remains to be demonstrated.

NADPH and ATP are produced in the light phase of photosynthesis. The next phase of photosynthesis involves the fixation of CO2 into carbohydrates. Although many textbooks state that glucose (C6H12O6) is the major product of photosynthesis, the actual carbohydrate endproducts are sucrose, paramylon, starch, etc. The fixation of CO2 takes place during the light independent phase using the assimilatory power of NADPH and ATP in the chloroplast stroma (eukaryotic algae) or in the cytoplasm (prokaryotic algae). The light independent reactions do not occur in the dark; rather they occur simultaneously with the light reactions. However, light is not directly involved. The light-independent reactions are commonly referred to as the Calvin Benson Bassham cycle (CBB cycle) after the pioneering work of its discoverers.

The first metabolite was a 3-carbon organic acid known as 3-phosphoglycerate (3-PG). For this reason, the pathway of carbon fixation in algae and most plants is referred to as C3 photosynthesis. As the first product was a C3 acid, Calvin hypothesized that the CO2 acceptor would be a C2 compound. However, no such C2 substrate was found. Rather, it was realized that the CO2 acceptor was a C5 compound, ribulose 1,5-bisphosphate (RuBP), and that the product of carboxylation was two molecules of 3-PG. This crucial insight allowed the pathway of carbon flow to be determined.

While the Calvin-Benson-Bassham cycle ( CBB ) cycle involves a total of 13 individual enzymatic reactions, only two enzymes are unique to this pathway: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphorybulokinase (PRK). All other enzymes involved also perform functions in heterotrophic metabolism. PRK catalyzes the phosphorylation of ribulose-monophosphate to ribulose-1,5- bisphosphate (RUBP). RUBP in turn is the substrate for RuBisCO, which catalyzes the actual carbon fixation reaction.

RuBisCO
As a result, the RuBisCO enzyme alone represents the most important pathway by which inorganic carbon enters the biosphere. . It is thought that as much as 95% of all carbon fixations by C3 organisms (that includes all phytoplankton) occur through RuBisCO.

RuBisCO is known to catalyze at least two reactions: the reductive carboxylation of ribulose 1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate and the oxygenation of RuBP to form one molecule of 3-PG and one molecule of 2-phosphoglycolate.

The oxygenation of RuBP is commonly referred to as photorespiration and has traditionally been seen as a wasteful process, in particular because the regeneration of RuBP in photorespiration leads to the evolution of CO2 and requires free energy in the form of ATP. 


Both reactions (carboxylation and oxygenation) occur in the same active site and compete, making the enzyme extremely sensitive to local partial pressures of CO2 and O2. RuBisCO makes up 20–50% of the protein in chloroplasts. It acts very slowly, catalyzing three molecules per second. This is comparable to 1000 per second typical for enzymatic reactions. Large quantities are needed to compensate for its slow speed.  Lastly, RuBisCO rarely performs its function at a maximum rate (Kmax), since the partial pressure of CO2 in the vicinity of the enzyme is often smaller than even its Michaelis-Menten half-saturation constant (Km). 

Three-dimensional structures of the RuBisCO enzyme are now known for a number of species, including Synechococcus and most recently the green alga Chlamydomonas reinhardtii. On the basis of these data and other studies it is now believed that the primary catalytic structure of RuBisCO is a dimer of two large subunits (L2). In form I RuBisCO four L2 dimers are cemented to form L8S8 hexadecameric superstructure whereby the major contacts between the L2 dimers are mediated by the small subunits. A Mg2+ cofactor as well as the carbamylation of Lys201 is required for the activity of the enzyme. A loop in the beta barrel and two other elements of the large subunit, one in the N and one in the C terminus of the protein form the active site in Synechococcus. Small subunits apparently do not contribute to the formation of the active site.

 4

1) http://www.plantphysiol.org/content/155/1/27.full#ref-44
2) http://www.bio-catalyst.com/most-important-enzyme-in-the-world/
3) http://www.nature.com/nature/journal/v463/n7278/full/463164a.html
4) http://www.genome.jp/kegg-bin/show_pathway?ko00710

In order to synthesize Rubisco, one of the most important proteins on earth, following cell parts, organelles, proteins, etc. are required

https://reasonandscience.catsboard.com/t1554-the-rubisco-enzymes-amazing-evidence-of-design#3899

Rubisco is the most important enzyme on the planet. virtually all the organic carbon in the biosphere derives ultimately from the carbon dioxide that this enzyme fixes from the atmosphere. Without it, advanced life would not be possible. And we would not be able to debate our origins. All inquiry and quest about if we are ultimately the result of a powerful creator, or just random natural chemical reactions and emerging properties of lifeless matter, if biodiversity is due to evolution, or an intelligent designer, is second to the inquiry of how Rubisco came to be.  Rubisco has a  complex structure, functioning and synthesis process, many cell compartments, enzymes, proteins, and pathways are involved and required to assemble it,  the unfinished sub-units require co and post-translational modifications, specific proteins that help like assembly robots in the manufacturing process, sophisticated pathways, and mechanisms of protein import and targeting in chloroplasts through large multi-protein translocon complexes in the stroma, and advanced protein communication and information systems. All this is of bewildering complexity, where dozens of individual interconnected and finely tuned parts are required, a web of interlocked extremely complex advanced molecular machines where if one is missing, nothing goes, that defy the intelligence of the best scientists for decades to find out their structure, mechanisms, and functions. Could all this be due to natural processes?

In order to synthesize Rubisco, following  parts not directly involved in Rubisco synthesis, but nonetheless Rubisco depends on them to work:

The cell as a whole.
Chloroplast envelope translocon  Tic - Toc protein supercomplexes ( only in plants, not in bacteria )
The information of the machinery to synthesize Tic - Toc complexes, and assembly processes.
The enzymes used in the Calvin Cycle

Parts directly involved in Rubisco function, activation, and  synthesis:

DNA
A variety of nucleus-encoded factors
Sequence-specific RNA-interacting regulatory proteins
Pentatricopeptide repeat proteins MRL1
mRNA
The information in DNA to make the subunits
The information for assembling the  holoenzymes and the whole Rubisco protein complex
The machinery to make ATP
The light-dependent process to make NADPH
The transcription and translation machinery
A transport, recognition and communication system to conduct the synthesized subunits through the chloroplast envelope
The translocon complexes in the plastid
Rubisco activase enzymes
Activation by carbamylation of the ε-amino group of active-site Lys201 by a CO2 molecule
Rubisco's magnesium ion
DNA and its information, mRNA and the machinery to make Chaperones
GroEL/GroES  Chaperones
RbcX assembly chaperone
N-terminal transit peptide  of the S-subunits ( only in plants, not in bacteria )
Stromal processing metalloprotease
Peptide deformylase enzymes
Methyltransferase enzymes
Chaperone BSDII
Chaperone systems containing DnaJ-, DnaK (Hsp70)
CA1P phosphatase

Could these parts, proteins enzymes, etc.  have evolved separately and gradually? What about the RbcX Assembly Chaperone, specifically used as an assembly tool of Rubisco? What about the barrel-shaped GroEL/GroES chaperonins which perform their function with extremely impressive simplicity and elegance, namely helping over 100 different proteins to get into their correct shape and form, essential for function? ( in our case, helping the Rubisco RbcL subunits to get their proper shape ) Or how do proponents of evolution explain how natural selection would have favored the emergence of Hsp70 chaperones,  central components of the cellular network,  proteins which assist a large variety of protein folding processes in the cell by the transient association of their substrate-binding domain with short hydrophobic peptide segments within their substrate proteins?  That is in our case, their function of which was to prevent a still-useless rubisco small subunit from folding outside the chloroplast?  They are made, used during the synthesis process, and once Rubisco assembly has finished, these enzymes are discarded. This is very much a factory-like production and assembly-line process, using fully automatized and programmed nano-robot like molecular machines, namely enzymes.  Most parts, if missing, render 1. the assembly of Rubisco impossible, and 2. Rubisco useless. Many parts, if missing render it not fully functional and defective.  Besides the enzymes that have used in other biological systems, there would be no reason to make them unless all other parts were there too, and the assembly instructions of Rubisco. As an analogy, if you had to make the implementation of a car factory, why would you make the assembly chain of a piston, if you do not have all the precise instructions to make 1. the car as a whole, and 2. the instructions of the precise shape and the materials required for the piston in particular, and how to mount it in the motor? That's precisely what happens in the cell.  Evolution has no consciousness, and no foresight nor intelligence. But precisely that is required for PLANNING and make of blueprints. I cannot create a machine, without the precise drawing and project information in advance, which is required to make 1. the assembly tools 2. the subparts 2. the whole machine.

Further issues:  Why would natural chemical reactions  produce  three enzymes that are uniquely used in  the Calvin cycle ( they are not used in any other metabolic reaction ),
that is Rubisco, sedoheptulose bisphosphatase , and Phosphoribulokinase , if they have function only in the Calvin Cycle?


Furthermore: Would not only a designer know why it would be necessary to produce a special sequence of chain reactions and the right order and sequence of the enzymes provoking the reactions, handing over each of the products to the next processing step ? and to get the end products, required for further metabolic reactions?

Following enzymes are required in the Light-independent reactions of the Calvin Cycle :

in the first stage:
1.Rubisco 
2.phosphoglycerate kinase
3.glyceraldehyde 3-phosphate dehydrogenase

in the second stage:
1.Triose phosphate isomerase Fructose 1,6-bisphosphatase
2.Aldolase
3.Transketolase
4.Sedoheptulose-bisphosphatase
5.Sedoheptulose-1,7-bisphosphatase
6.Ribose-5-phosphate isomerase
7.phosphopentose epimerase

and in the last stage:
1.phosphoribulokinase

So the whole pathway requires 11 enzymes, of which 3 are unique to the pathway.

But let's assume we would have all enzymes required for the Calvin cycle. These reactions take the products (ATP and NADPH) of light-dependent reactions and perform further chemical processes on them. So unless the light-dependent reactions of photosynthesis are in place, the Calvin cycle will not work.

Unless you have the cell to host the organelles, and the stroma, and the chloroplast, and all the parts to synthesize the molecular machines, nothing was done either.

So this is one more irreducible and interdependent complex nanomolecular factory, which requires the planning and programming of a designer, and all parts functioning right from the start in an interlocked complex manner.

We must observe that Darwin's proposed test, on at least one major interpretation, is less generous than it first appears: "If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down." For, as Shapiro acidly but aptly noted, on the defects in such an appeal to the bare possibility in defense of the RNA world hypothesis -- making a remark that, we observe, inadvertently also applies to his preferred metabolism first scenario -- that:

On the one side, you have an intelligent agency based system of the irreducible complexity of tight integrated, information-rich functional systems which have ready on hand energy directed for such, that routinely generate the sort of phenomenon being observed. And on the other side imagine a golfer, who has played a golf ball through a 12 hole course. Can you imagine that the ball could also play itself around the course in his absence? Of course, we could not discard, that natural forces, like wind, tornadoes or rains or storms could produce the same result, given enough time. the chances against it however are so immense, that the suggestion implies that the non-living world had an innate desire to get through the 12 hole course.

Catalysis, activation pathway  4

The main reaction catalysed by Rubisco (the addition of CO2 and H2O to RuBP to yield two molecules of 3PGA) involves multiple discrete steps and associated intermediates of variable stability



To be functional, Rubisco requires prior

- activation by carbamylation of the ε-amino group of active-site Lys201 by a CO2 molecule, which is distinct from the substrate-CO2.
- The carbamylated Lys201 is stabilized by the binding of magnesium ion to the carbamate.

 The carboxylation involves at least four, perhaps five discrete steps and at least three transition states;

- enolization of RuBP
- carboxylation of the 2,3-enediolate
- hydration of the resulting ketone, carbon–carbon scission
- stereospecific protonation of the resulting carboxylate of one of the product 3PGA

Several, if not all, of these steps involve acid–base chemistry.

Question : Why should unintelligent natural mechanisms be the best explanation for the emergence of these highly specific , regulated and coordinated  steps, which are essential for the activation of Rubisco's activity ?  As long as the enzyme does not provide the full and complete function of this process, nothing goes, and ultimatively, advanced life on earth would not be possible . This mechanism as all others in biology seem to be the result of a planning intelligence, which set it all up through power, will and information. 

Role of the magnesium ion

One of the key players in the reaction catalysed by Rubisco is the magnesium ion. Apart from the carbamylated Lys201 which provides a monodentate ligand, the magnesium ion is liganded by two (monodentate) carboxylate ligands provided by Asp203 and Glu204 and three water molecules .



The active site in carbamylated Rubisco with a reaction intermediate analogue (2CABP) bound across the α/β-barrel. Residues implicated in the reaction mechanism are highlighted.

RuBP replaces two of these water molecules. For the reaction to proceed, a tight control of the charge distribution around the metal ion is crucial and this presumably also includes residues outside the immediate co-ordination sphere. For instance, the metal ligands Asp203 and Glu204 interact with their free oxygen atom with the ε-amino groups of Lys175 and Lys177, respectively. These interactions may help avoid bidentate co-ordination of the carboxylate groups to the metal ion, which, if it occurred, would block the binding of the gaseous substrates.

CO2 replaces the last Mg2+-co-ordinated water molecule and adds to the enediol directly without forming a Michaelis complex. The resulting six-carbon compound, 3-keto-2′-carboxyarabinitol-1,5-bisphosphate is relatively stable. The six-carbon carboxylated intermediate is closely mimicked by the inhibitor 2′-carboxyarabinitol-1,5-bisphosphate , which forms an exchange-resistant complex. The crystal structures show Lys334 positioned to facilitate the addition of the gaseous substrate  in accord with site-directed mutagenesis and chemical modification.

Conformational changes during catalysis
During catalysis, Rubisco undergoes a conformational change, which serves to close the active site and prevent access of water during the reaction. The closing mechanism involves movements of loop 6 (a loop connecting β-strand 6 with α-helix 6 in the α/β-barrel), the carboxy-terminal strand, and a loop from the amino-terminal domain of the adjacent large subunit of the L2 dimer . Rubisco structures can be divided into two states ; open with the active site unliganded or occupied by loosely bound substrates or products, or closed with substrates or inhibitors tightly bound and completely shielded from solvent. Apart from the movement of loop 6 (residues 331–338) to cover the opening of the α/β-barrel, the transition between open and closed forms involves a rigid-body movement that brings the amino- and carboxy-terminal domains of adjacent subunits together, and the ordering of residues 63–69 of the amino-terminal domain. Packing of the carboxy-terminal strand (residues 463 to the carboxy-terminal end) against loop 6 completes the closure.

Two strictly conserved glycine residues, Gly333 and Gly337, maintain flexibility in the hinge of loop 6. The other strictly conserved residue, Lys334, is located at the tip of the loop and interacts with the incoming gaseous substrate during catalysis. The Lys334 side chain extends into the active site and hydrogen bonds to one of the two oxygen atoms of the inhibitor 2CABP that is equivalent to that of substrate CO2 . It also interacts with the γ-carboxylate of Glu60 and the hydroxyl group of Thr65 in the amino-terminal domain of the adjacent large subunit.

Mutations in loop 6 influence the CO2/O2 specificity
The importance of loop 6 for catalysis and specificity has been demonstrated by genetic selection and site-directed mutagenesis. Residue Val331 is part of the hinge on which the loop moves and is highly, but not strictly, conserved . Replacement of Val331 by Ala in the green alga Chlamydomonas reinhardtii  reduces specificity and carboxylation turnover.

The carboxy-terminus of the large subunit is important for proper loop closure
The interaction of the carboxy-terminus with loop 6 seems to be intimately involved in the transition from the open to the closed state of the Rubisco active site . As shown by site-directed mutagenesis, the carboxy-terminus is not absolutely required for catalysis, but is needed for maximal activity and stability . Residue Asp473 was proposed to serve as a latch responsible for placing the large-subunit carboxy-terminus over loop 6 and stabilizing the closed conformation required for catalysis . Directed mutagenesis and chloroplast transformation in C. reinhardtii showed that although Asp473 is not essential for catalysis, mutations D473A and D473E caused substantial decreases in catalytic efficiency and specificity . The crystal structure of D473E  showed that the relatively modest substitution of Asp473 by Glu causes disorder of the last six carboxy-terminal residues. It appears that the mutations disrupt contacts of residue 473 with Arg134 and His310. This may cause a destabilization of the underlying loop 6 with consequences for catalysis and specificity.

As shown, slight mutations cause severe compromise of the reaction, or even loss of function. This is a highly precise ordered complex process which only functions with all parts correctly in place being able to do the right processes, and if every part is doing its job correctly. Unless everything is in place, nothing goes. Thats a process definitively better explained through design, rather than random evolutionary processes without intelligence involved to set it up propperly.

Activation 2

The central role of RuBisCO in the fundamental process of photosynthesis means that it must be tightly regulated, to ensure it is active only where and when it should be. One important layer of this regulation is the activation of RuBisCO at the beginning of the day. During the night, the RuBisCO active sites are blocked by inhibitors or misfired reactants. These inhibitors include “daytime” substrates, such as RuBP, as well as specific inhibitors to ensure that RuBisCO is only active when there is a source of free energy from sunlight (otherwise, RuBisCO will use the free energy released by the breakdown of sugars via respiration, which constitutes a futile cycle – RuBisCO uses energy to fix carbon as sugars in the CBB cycle, then breaks these sugars down to re-release the energy so that it can fix more carbon!).

Activation, then, involves firstly the removal of these inhibitors. This is carried out by a light-activated chaperone protein imaginatively called RuBisCO activase. RuBisCO activase uses the free energy of ATP hydrolysis to “clear out” the active sites of RuBisCO.

The second step in activation of RuBisCO is called carbamylation. An activating CO2 molecule (note, not a substrate for catalysis) is added to a specific amino acid residue in the active site (Lysine201) . The carbamate thus formed is stabilised by a magnesium ion (Mg2+) – this is a further checkpoint to ensure RuBisCO is activated during periods of photosythetic activity, as the Mg2+ concentration of the chloroplast stroma increases dramatically during the day when Mg2+ floods out of the photosynthetically active thylakoids.

Following carbamylation, the substrate RuBP can bind to the active site, forming an enediol. C-terminal loops of RuBisCO now fold over the active site to create a channel down which the substrate CO2 (or O2, see below) molecule can diffuse. Catalysis then occurs when the CO2 (or O2) molecule binds to the enediol. RuBisCO is now active.

Regulation by RuBisCO activase


In plants and some algae, another enzyme, RuBisCO activase, is required to allow the rapid formation of the critical carbamate in the active site of RuBisCO. RuBisCO activase is required because the ribulose 1,5-bisphosphate (RuBP) substrate binds more strongly to the active sites lacking the carbamate and markedly slows down the "activation" process. In the light, RuBisCO activase promotes the release of the inhibitory, or — in some views — storage RuBP from the catalytic sites. Activase is also required in some plants  because, in darkness, RuBisCO is inhibited (or protected from hydrolysis) by a competitive inhibitor synthesized by these plants, a substrate analog 2-Carboxy-D-arabitinol 1-phosphate (CA1P). CA1P binds tightly to the active site of carbamylated RuBisCO and inhibits catalytic activity. In the light, RuBisCO activase also promotes the release of CA1P from the catalytic sites. After the CA1P is released from RuBisCO, it is rapidly converted to a non-inhibitory form by a light-activated CA1P-phosphatase. Finally, once every several hundred reactions, the normal reactions with carbon dioxide or oxygen are not completed, and other inhibitory substrate analogs are formed in the active site. Once again, RuBisCO activase can promote the release of these analogs from the catalytic sites and maintain the enzyme in a catalytically active form. The properties of activase limit the photosynthetic potential of plants at high temperatures. CA1P has also been shown to keep RuBisCO in a conformation that is protected from proteolysis. At high temperatures, RuBisCO activase aggregates and can no longer activate RuBisCO. This contributes to the decreased carboxylating capacity observed during heat stress.

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity 3

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants.  Rubisco activase forms a wide range of structures, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μM, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.

Structural mechanism of RuBisCO activation by carbamylation of the active site lysine 1

ABSTRACT

RuBisCO has been studied extensively by biochemical and structural methods; Following is the description of the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules.

Questions : How did the correct insertion of the correct metal cation Mg2+ surrounded by three H2O/OH molecules emerge ? Trial and error ? The genome needs the right information in order to get the right materials, how to position them at the right active site, and how to mount these parts in the right order . That seems to me only being explained in a compelling manner by a intelligent designer, which knew how to build this highly sophisticated and complex machine fully functional. A step wise emergence might not be completely impossible, but seems to be extremely unlikely.

Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) is a dominant contributor to conversion of gaseous CO2 into biomass . Aerobic life forms, including heterotrophs, primarily derive their carbon through an authotrophic/photosynthetic route using CO2 as a carbon source. RuBisCO accomplishes this task by incorporating CO2 into a phospho-sugar, ribulose 1,5-bisphosphate (RuBP). Incorporation of CO2 into RuBP generates two molecules of 3-phosphoglycerate. This simple compound is subsequently used to build other organic molecules of life. Synthesized RuBisCO does not have a fully functional active site. It needs to be activated by a CO2 molecule that carbamylates its catalytic Lys to bind Mg2+ that completes the activation process.



(A) General view of hexadecameric structure G. sulphuraria RuBisCO with three L2S2 dimers visible. The dimers on both sides are in a Cα representation colored by the temperature factors. The front-facing dimer is depicted in a surface representation colored by the polarity of the electrostatic field. Two large, positively charged patches (in blue) indicate the entry to the active site cavity that is lined with highly mobile loops with higher temperature factors coming from the β-barrel catalytic domains and the N-terminal domain (visible in dimers on both sides). These positively charged patches are visible because the disordered N- and C-terminal fragments were not included in our model (not visible in ED). They are responsible for sealing off the active sites during the catalytic cycle.

(B) A L2S2 dimer of G. partita RuBisCO in the same orientation as in A with the highlighted N-terminal (orange) and C-terminal (red) regions that are disordered in G. sulphuraria structure. (C) Close-up of the L1 subunit with N- and C-terminal tails marked and the loops numbered. Both termini play a crucial role in stabilizing close conformation of the catalytically competent enzyme. The C-terminal tail stabilizes loops 6, 7, and 5 whereas the N-terminal region stabilizes loop 1 (containing the KPK motif) and loops 2s and 3s of the small domain.

The most common form I is a L8S8 hexadecamer (Fig. above). This form is present mostly in chemoautotrophic bacteria, cyanobacteria, red and brown algae, and all higher plants. Form I RuBisCO has four subtypes: A and B from cyanobacteria, eukaryotic algae, and higher plants and C and D from nongreen algae and phototropic bacteria (red-type enzymes). The form II enzyme is an L2 dimer of large subunits only. Form III is diverse in composition (L2, L8, and L10) but does not contain small subunits. Form IV encompasses RuBisCO-like proteins from organisms that do not use CO2 as a major carbon source (20). The focus of this study is the L8S8 hexadecameric form ID of RuBisCO with a molecular weight of ∼0.6 MDa from red algae Galdieria sulphuraria .



The ribbon model of the central catalytic domain with bound gaseous ligands at the active site and the accessible surface colored by the electrostatic potential. The ligands are embedded in a positively charged cavity (blue) located at the C-terminal end of the β-barrel of the catalytic domain closed from the top with a negatively charged lid (red) formed by the N-terminal domain of the L subunit of the opposite dimer. Both of these features are responsible for a strong gradient of the electrostatic field across the active site that interacts with quadrupole moments of the gaseous ligands. The bound ligands are (A) dioxygen (in red) and (B) carbon dioxide (in purple).

This is a finely designed and tuned cavity to receive and host the molecules. Amazing.

The quadrupole moments of charge-less and dipole-less ligands interact only with the high electrostatic field gradient that provides a sufficient guiding force for discrimination and binding at the active site. This apparent need for a high gradient at the active site region explains why the minimal active form of RuBisCO is a dimer of large subunits. Unlike monomers, only dimers produce sufficiently strong electric field gradient across the active site by combining highly and oppositely charged domains that are required for the interaction of RuBisCO with the gaseous substrates.

Its evident that such a configuration could not arise is a gradual stepwise manner, since the catalytic site would become active only in the right, finely tuned configuration.

All of the factors described in this paper, i.e., existence of a strong electric field gradient that interacts with quadrupole moments of the substrates, temperature activation associated with protein mobility, and finally directional binding of the ligands, affect the efficiency of the enzyme.

That means, if they are not just right just from the start, nothing goes.....

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503183/
2) https://cambridgecapp.wordpress.com/improving-photosynthesis/rubisco/
3) http://www.jbc.org/content/288/28/20607.abstract
4) http://jxb.oxfordjournals.org/content/59/7/1555.full

Biogenesis of the Rubisco enzyme

Isolated pea chloroplasts are able to synthesize only a small proportion of their constituent proteins, such as the large subunit of Rubisco, RbcL. The remainder (including the small subunit of Rubisco, RbcS) are synthesized outside of the organelle on cytosolic ribosomes.

Advancing Our Understanding and Capacity to Engineer Nature’s CO2-Sequestering Enzyme, Rubisco1 1



Hypothetical profiles of Rubisco phylogeny, the evolutionary timelines of different photosynthetic organisms, and variation in atmospheric CO2 (thicker line) and O2 levels during earth’s history. Hypothetical atmospheric CO2 and O2 levels prior to 0.6 billion years ago are represented by dotted lines. Quaternary structures of each Rubisco were drawn with Pymol using Protein Data Bank coordinates for the spinach (Spinacia oleracea) (L2)4S8 (8RUC), R. rubrum L2 (5RUB), Pyrococcus horikoshii (L2)4 (2CWX), and Thermococcus kodakaraensis (L2)5 (1GEH) enzymes. Structures for larger form II (L2)n Rubiscos are unavailable. Circular images depict types of organisms where the different Rubisco forms are found.

RUBISCO AND ITS INTERACTIONS WITH OTHER PROTEINS

Rubisco Expression and Assembly

In prokaryotes and the plastid genome of nongreen algae, genes for the L- (rbcL) and S- (rbcS) subunits of form I Rubiscos are colocated in an operon. In higher plants and green algae, the single rbcL gene remains encoded by the plastid genome, while multiple copies of the RbcS gene are located in the nucleus (Fig. below). The process(es) by which expression of the plastid- and nucleus-encoded L- and S-subunits, respectively, are coordinated remains unclear. Recent evidence suggests that L-subunit expression may be controlled by the epistasy of synthesis (CES) paradigm, wherein unassembled L-subunit motifs bind to the rbcL mRNA to autoregulate its translation. The importance of regulating Rubisco synthesis in plastids is paramount, as it is produced in high quantities to account for its slow and unspecific enzymatic activity. For example, in C3 plants, between 20% and 30% of the leaf protein (i.e. approximately 25% of the leaf nitrogen) is invested in Rubisco.




The complexity of Rubisco biogenesis and its regulation by RA in vascular plant chloroplasts. Putative and known Rubisco-specific processes and interacting molecular partners (coded by genes in the nucleus as shown) are highlighted in white rectangles. See text for details of the processes, abbreviations, and the challenges faced in modifying the L8S8 enzyme. Uncertainties in the biogenesis process are indicated by question marks. Hsp, Heat shock proteins; PEP, plastid-encoded RNA polymerase proteins; Pep, unknown Met-1, Ser-2 peptidase activity; Rca, nucleus gene coding RA; TAFs, nucleus-encoded trans-acting factors, Tic and Toc, chloroplast translocon inner and outer membrane complexes respectively; 30S/50S and 40S/60S, stromal and cytosolic ribosomal subunits.

As with other plastid-localized proteins, synthesis of the S-subunit in the cytosol necessitates an appropriate N-terminal transit peptide for transfer (with the aid of molecular chaperones Hsp70)  10   to, and then passage through, the chloroplast envelope translocon complexes . Within the stroma, the S-subunits undergo further posttranslational modification (transit peptide cleavage, Met-1 αN-methylation) prior to assembly into L8S8 complexes.


Folding and assembly of RuBisCO and the role of chaperones  8

Prokaryotic organisms synthesize and assemble their RuBisCO in the cytosol after transcription of a single gene for large subunits (type II RuBisCO, RbcL2) or of an operon encoding both the large and small subunits (type I RuBisCO, RbcL8S8). In eukaryotic cells, functional type I RuBisCO is only found in the chloroplasts. However, the small subunit rbcS genes are generally encoded in the nucleus as a small multi-gene family of up to more than ten members. Therefore, after translation on cytosolic ribosomes, the small subunit precursor proteins have to be imported into the chloroplasts, where they are processed and fold to the native state. In contrast, higher plants` large subunit rbcL genes occur as a single copy per chloroplast genome, but since each chloroplast contains several copies of this genome, a higher number of the same rbcL gene can be found in each chloroplast. These rbcL genes are structurally similar to prokaryotic genes (and therefore also amenable to expression in E.coli). For example, they lack introns, have 5`-sequences that resemble bacterial promotors and produce mRNAs with Shine-Dalgarno-like sequence stretches. Moreover, their translation is initiated by fmet-tRNA and carried out by 70S ribosomes in the chloroplast stroma .

The chaperonin Cpn60/GroEL is one fundamental factor in the folding and assembly pathway of RuBisCO.

In the current view of the RbcL8S8 holoenzyme maturation pathway, large subunits are folded by and released from Cpn60 in a manner dependent on Mg2+ and ATP, followed by the formation of RbcL2 dimers. The latter subsequently tetramerize around a fourfold axis to RbcL8 cores, before eight small subunits spontaneously associate with the RbcL8 core on top and bottom, resulting in RbcL8S8

Biogenesis of L-subunits in the chloroplast begins with the transcription of rbcL via a plastid-encoded RNA polymerase that requires a variety of nucleus-encoded factors , including sequence-specific RNA-interacting regulatory proteins. A conserved pentatricopeptide repeat protein, MRL1, has recently been identified in Chlamydomonas and Arabidopsis (Arabidopsis thaliana) that specifically binds to the 5′ untranslated region of rbcL to stabilize the mRNA and/or ensure correct processing of the transcript . The fundamental aspects of rbcL translation and posttranslational processing in chloroplasts also lack detail but appear to show similarities to the bacterial translational machinery , albeit reliant on nucleus-encoded factors for proper functioning . Nascent L-subunits are targeted for extensive N-terminal processing  that begins with the deformylation of N-formyl-Met-1 by peptide deformylase and then Met-1 and Ser-2 removal via an uncertain peptidase process, leaving an N-terminal Pro-3 that is acetylated by an unknown αN-acetyltransferase. In some species, Lys-14 is also trimethylated by a Rubisco L-subunit εN-methyltransferase. Recent single-particle cryoelectron microscopy analysis revealed a large contact area between Rubisco L-subunit εN-methyltransferase and the C- and N-terminal domains of L-subunit pairs in L8S8 Rubisco but not the S-subunits, suggesting that trimethylation of Lys-14 occurs after L2 assembly  and prior to S-subunit assembly. Although the functions of these posttranslational modifications remain unclear, it is assumed that they protect plant Rubisco from proteolytic degradation.

In plastids, it is thought that newly translated L-subunits interact with the general Hsp70 chaperone system (DnaK/DnaJ/GrpE) and the Rubisco-specific chaperone BSDII. These chaperones prevent misfolding and convey the unfolded L-peptide to the folding cage of the chaperonin-60/21 complexes (plant homologous of the GroEL and GroES Escherichia coli proteins). Studies with cyanobacteria L8S8 Rubisco have shown that chaperonin-folded L-subunits interact with RbcX, a Rubisco-specific chaperone whose gene (rbcX) is often located between rbcL and rbcS in cyanobacteria. RbcX dimers facilitate the assembly of L-subunits into (L2)4 complexes and are then displaced by the stable binding of S-subunits that produce the native L8S8 enzyme. In Synechococcus PCC7942, where rbcX is located separate from the rbcL-rbcS operon, deletion of rbcX has no effect on Rubisco synthesis. This begs the question of whether the nucleus-encoded RbcX homolog(s) in higher plants has a functional role in Rubisco assembly in plastids.



To become biologically active, proteins have to acquire their correct three-dimensional structure by folding, which is frequently followed by assembly into oligomeric complexes. Although all structure relevant information is contained in the amino acid sequence of a polypeptide, numerous proteins require the assistance of molecular chaperones which prevent the aggregation and promote the efficient folding and/or assembly of newly-synthesized proteins. RuBisCO requires chaperones in order to acquire its active structure. In plants and cyanobacteria, RuBisCO (type I) is a complex composed of eight large (RbcL) and eight small (RbcS) subunits.

It is known that folding of RbcL is accomplished by chaperonin and most likely supported by the Hsp70 system, whereas recent findings indicate the additional need of specific chaperones for assembly. 

The major obstacle for reconstitution was found to be the incapability to produce RbcL8 cores competent to form RbcL8S8 holoenzyme. It could be shown that the RbcL subunits interact properly with the chaperonin GroEL in terms of binding, encapsulation and cycling. However, they are not released from GroEL in an assembly-competent state, leading to the conclusion that a yet undefined condition or (assembly) factor is required to shift the reaction equilibrium from GroEL-bound RbcL to properly folded and released RbcL assembling to RbcL8 and RbcL8S8, respectively.

Cyanobacterial RbcX was found to promote the production of cynanobacterial RbcL8 core complexes downstream of chaperonin-assisted RbcL folding, both in E. coli and in an in vitro translation system. Structural and functional analysis defined RbcX as a homodimeric, arc-shaped complex , which interacts with RbcL via two distinct but cooperating binding regions. A central hydrophobic groove recognizes and binds a specific motif in the exposed C-terminus of unassembled RbcL, thereby preventing the latter from uncontrolled misassembly and establishing further contacts with the polar peripheral surface of RbcX. These interactions allow optimal positioning and interconnection of the RbcL subunits, resulting in efficient assembly of RbcL8 core complexes. As a result of the highly dynamic RbcL-RbcX interaction, RbcS can displace RbcX from the core-complexes to produce active RbcL8S8 holoenzyme. Species-specific co-evolution of RbcX with RbcL and RbcS accounts for limited interspecies exchangeability of RbcX and for RbcX-supported or -dependent assembly modes, respectively.
.


RbcX functions to increase the efficiency of Rubisco assembly by acting on folded RbcL subunits subsequent to their GroEL/GroES-mediated folding. Recognition of RbcX requires the exposed C-terminal RbcL peptide (see Discussion for details and Figure S11). Note that assembly of RbcL8S8 may also occur independently of RbcX for some Rubisco homologs, presumably involving similar assembly intermediates

Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco 6

Molecular chaperones fulfill essential functions in protein biogenesis and quality control in all types of cell . 7 Although the term “molecular chaperone” was coined to describe a role in assisting oligomeric protein assembly , it is now commonly thought that chaperones are primarily involved with mediating polypeptide chain folding, essentially by preventing misfolding and aggregation of newly-synthesized and stress-denatured polypeptides.

These mechanisms are well established, particularly for the Hsp70s and the chaperonins . By contrast, how cells ensure the efficient assembly of folded subunits into oligomeric complexes is not well understood, and surprisingly few examples of specific assembly chaperones are known . Hexadecameric ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in chloroplasts, the most abundant enzyme in nature, has been an important paradigm in studies of protein assembly. Indeed, this process was initially thought to be mediated by the chaperonin system because nonassembled large subunits of plant Rubisco were observed to cofractionate with the chloroplast chaperonin . However, attempts to reconstitute Rubisco with chaperonin alone have failed, suggesting that additional factors must participate in this process.

Rubisco catalyzes the initial steps of two opposing reaction pathways: photosynthetic carbon fixation (CO2 as the substrate) and photorespiration (O2 as the substrate. The photosynthetic fixation of CO2 results in the synthesis of usable sugars  and thus is responsible for plant growth and yield.

Three structural forms of assembly of otherwise closely homologous Rubisco subunits are known :

Form I found in plants and cyanobacteria is a hexadecamer, denoted as RbcL8S8, containing eight large subunits  and eight small subunits . The oligomer has a cylindrical shape with a diameter . Four small subunits cap the top and bottom of eight large subunits, which form a tetramer of dimers.

The simpler form II Rubisco in some photosynthetic bacteria (e.g., Rhodospirillum rubrum) and dinoflagellates is a dimer of RbcL subunits.

Form III, found in a thermophilic archaeon, consists of five RbcL dimers.

Chaperonins are large cylindrical complexes with ATPaseactivity. The GroEL-type chaperonins (Cpn60s) of bacteria, chloroplasts, and mitochondria consist of two heptameric rings , each forming a central cavity for the binding of nonnative protein substrate . They function together with factors of the GroES/Cpn10 family, single-ring assemblies of seven  subunits, which bind transiently to the ends of the chaperonin cylinder. ATP-dependent GroES association results in the encapsulation of a single molecule of unfolded protein inside the GroEL cavity for folding to occur unimpaired by aggregation. Whereas bacterial GroEL and the mitochondrial chaperonin are homo-oligomeric, the chloroplast chaperonin is composed of homologous α and β subunits. Moreover, plant chloroplasts have two types of cochaperone, Cpn10 and Cpn20, the latter consisting of a tandem repeat of Cpn10 units. These features may represent an adaptation to chloroplast-specific substrate proteins.

The folding and assembly of form II Rubisco from R. rubrum has been reconstituted with bacterial GroEL/GroES . In this reaction Rubisco subunits fold inside the chaperonin cage, followed by dimerization upon release into solution . Expression of cyanobacterial form I Rubisco of Synechococcus sp. PCC6301 (also known as Anacystis nidulans) in E. coli was reported to result in the chaperonin-dependent production of enzymatically active holoenzyme. As shown in cyanobacteria, assembly of the RbcL8S8 complex is thought to involve the formation of RbcL8 core particles, followed by the docking of unassembled small subunits . Recently it has been reported that the product of the rbcX gene, present in the intergenic space between the rbcL and rbcS genes in several cyanobacteria, enhances the production of enzymatically active Rubisco upon coexpression with rbcL and rbcS in E. coli . Partial inactivation of rbcX in Synechococcus sp. PCC7002 resulted in a substantial reduction in Rubisco solubility and activity . However, the rbcX gene was reported to be nonessential in Synechococcus sp. PCC7942 .

Here we report that cyanobacterial RbcX is a Rubisco assembly chaperone. Structural and mechanistic analysis of RbcX from Synechococcus sp. PCC7002 revealed that the protein functions as a homodimer by binding and stabilizing RbcL subunits subsequent to their interaction with chaperonin. RbcX recognizes specifically, via a central peptide-binding groove, a conserved C-terminal peptide of RbcL. This interaction and additional contacts mediated by a peripheral binding surface of RbcX assist in the efficient formation of RbcL8 core complexes. The RbcL-RbcX interaction is dynamic, facilitating displacement of RbcX from RbcL8 by RbcS subunits to produce the active holoenzyme. The interchangeability of RbcX proteins between species is limited, suggesting that RbcX coevolved with RbcL and RbcS. We find that RbcX homologs are also present in higher plants.

Crystal structure of a chaperone-bound assembly intermediate of form I Rubisco 3

The form I Rubisco of autotrophic bacteria, algae and plants is a complex of eight large (RbcL) and eight small (RbcS) subunits. It fixes atmospheric CO2 in the dark reaction of photosynthesis. As shown for the cyanobacterial enzyme, folding of the RbcL subunits is mediated by the GroEL–GroES chaperonin system, and assembly requires the specialized chaperone RbcX, a homodimer of ~15-kDa subunits. Here we present the 3.2-Å crystal structure of a Rubisco assembly intermediate, consisting of the RbcL8 core with eight RbcX2 molecules bound. The structure reveals the molecular mechanism by which RbcX2 mediates oligomeric assembly. Specifically, RbcX2 provides positional information for proper formation of antiparallel RbcL dimers, thereby preventing RbcL–RbcL misalignment and off-pathway aggregation. The RbcL8(RbcX2)8 structure also suggests that RbcS functions by stabilizing the '60s loop' of RbcL in the catalytically active conformation.

Unless RbcL–RbcL are not properly aligned and aggregated, Rubisco is not able to become active to exercize its funtion. Question : For what reason would RbcX assembly enzymes evolve, if there was no forsight and planning of unconscous matter in regard of what the final function would be ? These helper enzymes are highly specific in their form, function and aggregation. Nobody would build a programmed robot for a assembly line, if the final purpose were not known right from the beginning......  



Upon folding and release from the GroEL–GroES complex, the RbcL subunit is recognized by RbcX2, which binds the flexible C-terminal RbcL peptide (area I) as well as area II on the folded body of the subunit (step 1).

The antiparallel RbcL dimer then forms (step 2)

the complementary surface charges on both RbcL and RbcX2 are likely to have a role in guiding dimer formation, avoiding misalignment. The RbcX2 molecules, each contacting area III on the respective adjacent RbcL subunit, function as 'molecular staples' in stabilizing the dimer. The stable RbcL2(RbcX2)2 units subsequently assemble to the RbcL8 core complex (step 3)

In this complex, a large portion of the RbcS-binding interface is preformed. RbcS binding structures the RbcL N terminus and the 60s loop, which sterically blocks access of RbcX2 to binding area III on RbcL. This facilitates displacement of RbcX2 and formation of the functional Rubisco holoenzyme (step 4)



(a) Close-up view showing surfaces on the antiparallel dimer of RbcL that interact with RbcX2. The outline of the bound RbcX2 is shown for orientation. The interaction surfaces area 1 (purple) and area II (cyan) are located on one RbcL subunit, whereas area III (red) is located on the adjacent subunit of the RbcL dimer.

(b) Ribbon diagram of the RbcX dimer showing the contact regions with RbcL, colored as in a. RbcX2 is rotated 180° relative to the view shown in a.

(c) Close-up view of the RbcX2 interface with the C-terminal peptide of RbcL (area I). RbcX2 is shown in surface representation; interface residues are indicated. The C-terminal peptide of RbcL is shown in stick model. In the background, the area II contact between loop 6 of RbcL and residue Gln5 of one RbcX chain (green) is visible. Oxygen and nitrogen heteroatoms are indicated in red and blue, respectively.

(d) Cutaway view of the RbcX2 interface with the opposing RbcL subunit (area III). The surface of RbcX2 is shown as a transparent skin. Crucial contact residues in RbcX and RbcL are shown in stick representation. The β-sheet in the N-terminal domain of RbcL, which would be in the foreground, is omitted for clarity.



Role of auxiliary proteins in Rubisco biogenesis and function 2

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the conversion of atmospheric CO2 into organic compounds during photosynthesis.  Much has been learnt about the complex cellular machinery involved in Rubisco assembly and metabolic repair over recent years. The simple form of Rubisco found in certain bacteria and dinoflagellates comprises two large subunits, and generally requires the chaperonin system for folding. The hexadecameric Rubisco, which comprises eight large and eight small subunits, from its dimeric precursor has rendered Rubisco in most plants, algae, cyanobacteria and proteobacteria dependent on an array of additional factors. These auxiliary factors include several chaperones for assembly as well as ATPases of the AAA+ family for functional maintenance.

 2



Schematic representation of photosynthesis in chloroplasts. The light reaction and the Calvin–Benson–Bassham cycle of CO2 fixation, as well as the side-reaction of photorespiration are shown.

The form I Rubisco of autotrophic bacteria, algae and plants is a complex of eight large (RbcL) and eight small (RbcS) subunits. It fixes atmospheric CO2 in the dark reaction of photosynthesis.  Folding of the RbcL subunits is mediated by the GroEL–GroES chaperonin system see figure below:




a, Structure of the GroEL tetradecamer complex (PDB: 1GRL) and the GroES heptamer (PDB: 1AON) in surface representation. Two subunits of GroEL in opposite rings and one subunit in GroES are highlighted in ribbon representation.

b, Single subunit of GroEL in ribbon representation, indicating the apical, intermediate and equatorial domains. The grey spheres represent hydrophobic residues that point towards the central cavity of the GroEL cylinder and mediate binding of non-native substrate protein.

c, The generic GroEL–GroES reaction cycle. Substrate protein as a collapsed folding intermediate is bound by the open GroEL ring of the asymmetrical GroEL–GroES complex. Binding of ATP to the substrate-bound ring causes a conformational change in the apical domains which results in the exposure of the GroES binding residues. Binding of GroES causes substrate displacement into an enclosed folding cage. ADP and GroES dissociate from the opposite ring together with previously-bound substrate (not shown). The newly-encapsulated substrate is free to fold in the GroEL cavity during the time needed to hydrolyse the bound ATP molecules (∼2–7 s dependent on temperature). ATP binding followed by GroES binding to the opposite ring triggers GroES-dissociation, releasing the substrate protein.

and assembly requires the specialized chaperone RbcX. ( see figure below: )



a, Structure of the RbcX dimer from the cyanobacterial species Syn7002 (PDB: 2PEI). Protomers are shown in ribbon representation.

b, Surface conservation of RbcX. The similarity score from an alignment of 151 sequences of cyanobacterial RbcX in the PFAM database was plotted onto the accessible surface of the RbcX dimer. Sequence conservation is displayed as a color gradient, indicating highly conserved residues in magenta and variable regions in cyan. The positions of conserved surface residues are indicated. 

c, Peptide binding cleft of RbcX. The C-terminal peptide EIKFEFD of RbcL, shown in ball-and-stick representation, is bound in the central cleft of the RbcX, shown in surface representation (PDB: 2PEM). The positions of residues in RbcX critical for peptide binding as well as the critical residues of the peptide are indicated. N- and C-termini of the peptide are also indicated. 

d, Overall architecture and dimensions of the RbcL8RbcX8 assembly intermediate. The RbcL8 core structure of Syn6301 is shown in surface representation and the bound RbcX in ribbon representation. The C-terminal tails of RbcL subunits are bound within the central cleft of RbcX and the conserved corner residues of RbcX are in contact with the N-domain of the adjacent RbcL subunit in the antiparallel dimer.

Here we present the  structure of a Rubisco assembly intermediate, consisting of the RbcL8 core with eight RbcX2 molecules bound. The structure reveals the molecular mechanism by which RbcX2 mediates oligomeric assembly. Specifically, RbcX2 provides positional information for proper formation of antiparallel RbcL dimers, thereby preventing RbcL–RbcL misalignment and off-pathway aggregation. The RbcL8(RbcX2)8 structure also suggests that RbcS functions by stabilizing the '60s loop' of RbcL in the catalytically active conformation.



a, Role of GroEL–GroES chaperonin and RbcX in green-type Rubisco assembly  and available structural information. Upon folding and release from the chaperonin complex, the cyanobacterial RbcL subunit is recognized by RbcX, which binds the flexible C-terminal RbcL peptide. Formation of the antiparallel RbcL dimer occurs mediated by two RbcX acting as molecular staples. The stable RbcL2RbcX2 units subsequently assemble to the RbcL8RbcX8 complex, in which a large portion of the RbcS binding interface is pre-formed. RbcS binding structures the RbcL N-terminus and the 60s loop, causing displacement of RbcX and formation of the functional Rubisco holoenzyme. Note that additional assembly factors, such as Raf1, are also involved in assembly but their mechanism of action remains to be determined. Figure modified from ref. 50, NPG.


 b,Role of RbcS in red-type Rubisco assembly based on mutational analyses and in vitro reconstitution 64. The RbcS subunit arrangement in the crystal structure of the red-type Rubisco from Alcaligenes eutrophus (PDB: 1BXN) is shown with the RbcS subunits in ribbon representation (red) and the RbcL octameric core in surface representation. The side view is a cross-section along the four-fold axis through the complex showing the central barrel formed by the β-hairpin extensions (see Supplementary Fig. 1) of the RbcS subunits which mediate assembly. SC, solvent channel. Figure modified from ref. 64, ASBMB.

Rubisco in complex with Rubisco large subunit methyltransferase 9

Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco

The large subunit (LS)1 is encoded by a plastid gene and is translated and assembled into Rubisco holoenzyme within the stroma of the chloroplast.  the LS is processed to the mature form by removal of the N-terminal Met-1 and Ser-2 residues and acetylation of Pro-3. The small subunit (SS) of Rubisco is also post-translationally modified. The polypeptide is post-translationally imported into chloroplasts and processed by a stromal processing peptidase that removes the targeting presequence. The resultant N-terminal
methionine residue of the processed SS is subjected to N-methylation  prior to assembly with the LS into the holoenzyme.




Docking of the crystal structures into the EM density map. (A and B) The crystal structures of Rubisco and RLSMT were fit into the 3D reconstruction of the Rubisco–RLSMT complex. Rubisco large and small subunits are dark and light green, respectively. RLSMT is red. The C terminus of RLSMT protrudes from the density map, but it may be folded back toward the C lobe of the protein as modeled in Fig. S4. (Scale bar, 5 nm.) (C) A detailed view of the Rubisco–RLSMT interface at the catalytic site. The RLSMT nSET, iSET, cSET, and SET regions are magenta, blue, orange, and cyan, respectively. The C-terminal lobe is shown in red, and the domain-swapped C-terminal extension in gold. The Rubisco substrate Lys-14 and the invariant active site Tyr-287 in RLSMT are shown in yellow and red ball-and-stick representations, respectively. RLSMT residues involved in substrate binding are shown in gray ball-and-stick representation.






1) http://www.plantphysiol.org/content/155/1/27.full
2) http://www.nature.com/articles/nplants201565#f1
3) http://www.nature.com/nsmb/journal/v18/n8/fig_tab/nsmb.2090_F6.html
4) http://edoc.ub.uni-muenchen.de/7577/
6) http://www.sciencedirect.com/science/article/pii/S0092867407005314
7) http://reasonandscience.heavenforum.org/t1437-chaperones-proteins-in-our-bodies-are-helped-by-other-proteins-known-as-chaperones-to-become-functional#2085
8 ) http://edoc.ub.uni-muenchen.de/7577/1/Saschenbrecker_Sandra.pdf
9) http://www.pnas.org/content/106/9/3160.full
10 ) http://reasonandscience.heavenforum.org/t1437-chaperones#2410

http://www.cell.com/cell/pdf/S0092-8674(07)00531-4.pdf
http://rstb.royalsocietypublishing.org/content/363/1504/2629.full

Co- and post-translational modifications in Rubisco 2

Both the large (LS) and small (SS) subunits of Rubisco are subject to a plethora of co- and post-translational modifications. With the exceptions of LS carbamylation and SS transit sequence processing, the remaining modifications, including


Deformylation
Nascent L-subunits are targeted for extensive N-terminal processing  that begins with the deformylation of N-formyl-Met-1 by peptide deformylase and then Met-1 and Ser-2 removal via an uncertain peptidase process, leaving an N-terminal Pro-3 that is acetylated by an unknown αN-acetyltransferase. In some species, Lys-14 is also trimethylated by a Rubisco L-subunit εN-methyltransferase. Recent single-particle cryoelectron microscopy analysis revealed a large contact area between Rubisco L-subunit εN-methyltransferase and the C- and N-terminal domains of L-subunit pairs in L8S8 Rubisco but not the S-subunits , suggesting that trimethylation of Lys-14 occurs after L2 assembly  and prior to S-subunit assembly . Although the functions of these posttranslational modifications remain unclear, it is assumed that they protect plant Rubisco from proteolytic degradation 1

acetylation, methylation, and N-terminal proteolytic processing of the LS, are still biochemically and/or functionally undefined although they are found in nearly all forms of Rubisco from vascular plants. A collection of relatively unique enzymes catalyse these modifications, and several have been characterized in other organisms. Some of the observed modifications in the LS and SS clearly suggest novel changes in enzyme specificity and/or activity

( that means they are used only for Rubisco processing )

and others have common features with other co- and post-translationally modifying enzymes. With the possible exception of Lys14 methylation in the LS, processing of both the LS and SS of Rubisco is by default an ordered process sequentially leading up to the final forms observed in the holoenzyme

( how did this ordered process emerge ? Had the process not have to be ordered in the way it is right from the beginning , otherwise the end product would not be synthesized ? )

An overview of the nature of structural modifications in the LS and SS of Rubisco is presented, and, where possible, the nature of the enzymes catalysing these modifications (either through similarity with other known enzymes or through direct enzymological characterization) is described. Overall, there are a distinct lack of functional and mechanistic observations for modifications in Rubisco and thus represent many potentially productive avenues for research.

Since its discovery as the principle enzyme responsible for photosynthetic fixation of carbon dioxide, a plethora of co- and post-translational protein modifications have been reported for Rubisco. Early findings describing proteolytic processing of the small subunit (SS)  and carbamylation 3 of the large subunit (LS) sparked the development of several avenues of research which continue today. While the aforementioned modifications were quickly established as essential for critical aspects of Rubisco assembly and activity, many others that followed have not been resolved with regard to functional significance. Nevertheless, Rubisco has served as an excellent model for many aspects of co- and post-translational processing that have resulted in significant contributions to our understanding of the biochemical processes and enzymes responsible for these modifications.

Here an overview is presented of the co- and post-translational modifications in both the LS and SS that are currently known primarily for the vascular plant forms of Rubisco but for which information about the enzymes catalysing these modifications and/or their functional significance is limited.

Colour-coded representation of the various post-translational modifications that occur on the LS (A) and SS (B) of Rubisco. Amino acids that are modified and/or removed during processing are highlighted in colours other than green. The enzyme and corresponding EC number is indicated alongside the arrow of the reaction it catalyses; a question mark is suggestive of the unknown nature of the enzyme. The dotted arrow denotes the species specificity of Rubisco LSMT activity. 

1) http://www.plantphysiol.org/content/155/1/27.full
2) http://jxb.oxfordjournals.org/content/59/7/1635.abstract
3) http://reasonandscience.heavenforum.org/t1554-the-rubisco-enzyme#3903
4) http://www.plantphysiol.org/content/155/1/27.full

Evolution cannot account for the assembly and activation of rubisco. 1

All attempts to reconstitute a 16-unit rubisco from any source have failed, so the assembly of rubisco must be studied in the chloroplast extracts. The eight large (L) subunits of rubisco are coded by the chloroplast DNA, and the eight small (S) subunits by nuclear DNA. The S subunit of rubisco is synthesized on free cytosolic polyribosomes* and maintained even during synthesis in an unfolded state by chaperones* of the Hsp70 class and their protein partners. When the small unit is brought to the import complex of the chloroplast, the fourteen-polypeptide chloroplast Cpn60 chaperonin protein associates with IAP100 (protein) of the import complex and can also associate with mature imported small subunits. The chloroplast Cpn60 chaperone is similar to the E. coli GroEl protein. After the unfolded precursor protein enters the stromal space, it binds briefly to a stromal Hsp70 chaperone protein and the N terminal targeting sequence is cleaved.

The large subunits of the rubisco enzyme are produced by the DNA and machinery of the chloroplast itself and stored complexed to a Cpn60 chaperonin. This chaperone protein keeps the large subunit protein from folding incorrectly, and therefore becoming useless, and is also necessary for the proper binding of the eight large subunits; without it they will form a useless clump.In many plants, the large subunits are chemically modified by specialized enzymes before they bind to the chaperonin protein. There is strong evidence that chloroplast Cpn60, Cpn21 and Hsp70 also participate in the assembly of the sixteen-unit rubisco complex. After a soluble L8 core is formed with the assistance of the chaperonin proteins, tetramers (four-part complexes) of small subunits bind to the top and bottom of the complex to form the complete enzyme. There are almost certainly other chaperones and chaperone-like polypeptides or lipo-proteins involved that are not yet characterized.

How do proponents of evolution explain how natural selection would have favoured a protein complex the function of which was to prevent a still-useless rubisco small subunit from folding outside the chloroplast? Before it evolved a way to get the protein inside, there would be no benefit from keeping it unfolded outside. How could blind chance ‘know’ it needed to cause large subunit polypeptides to fold ‘correctly’ and to keep them from clumping? It could not ‘anticipate’ the ‘correct’ conformation before the protein became useful. And evolution would need to be clever indeed to chemically modify something not yet useful so that it could be folded ‘correctly’ when even the ‘correctly’ folded polypeptide would not yet become useful.

Only a designer would know why it would be necessary to produce a specialized protease, target it to the chloroplast, and program it to clip off the targeting sequence of the small subunit at just the right place. And what about the assembly of a collection of meaningless rubisco parts in just one certain way? In order to design a sophisticated set of tools to make something else useful in the future that had, as yet, no function, evolution (as ‘designer’) would have had to have detailed knowledge of the future usefulness of the protein it was so cleverly engineering. If evolution managed to generate any one of these chaperone protein complexes (and it would not), it would still be useless for generating rubisco unless all the other chaperones were also present. Without any one of them, the sixteen-unit complex could not be generated.

But let us assume the impossible, that evolution succeeded in producing the rubisco enzyme complex, and that random chance happened to generate a new, otherwise useless, enzyme to create its substrate, RUBP. The perfect and complete rubisco sixteen-unit protein complex would then bind tightly to RUBP and do nothing.

In the real world, far away from the never-never land of evolution, another enzyme is needed to separate rubisco from RUBP. Once the rubisco complex is produced, a protein activase uses ATP energy to separate it from RUBP, to which it is tightly bound in its inactive (dark conditions) form. Apparently, the hydrolysis of ATP changes the configuration of the activase protein so that it can bind to rubisco and cause it to release its RUBP. The rubisco must then be carbamylated on the ε-NH2 group of just a certain lysine amino acid residue, and then it must pick up a Mg2+ ion on that carbamyl group* to form the active rubisco site. The amide group starts out as NH3+, which must become NH2 before the CO2 can be added, and another proton is lost when the COO- actually attaches, so that these steps are stimulated by low H+ concentration and high Mg2+. Light lowers the H+ concentration of the stroma by a process we have discussed, and raises the Mg2+ also. However, no RUBP can be detected in photosynthetic tissue at night, signifying that it is actually phosphoribulokinase that disrupts the cycle at night. What all of this implies is that even if evolution managed the impossible task of generating the rubisco enzyme, the entire system as it presently stands would be needed to turn it on in the light and off in the dark.

1) http://www.answersingenesis.org/articles/tj/v17/n3/photosynthesis

Rubisco “Highly Tuned” for Fixing Atmospheric Carbon 1

Rubisco sounds like a brand of cracker or something, but it’s actually an air cleaner your life depends on.  It’s an enzyme that fixes atmospheric carbon for use by photosynthetic microbes and plants.  In doing so, it sweeps the planet of excess carbon dioxide – the greenhouse gas implicated in discussions of global warming – making it a politically important molecule as well the most economically important enzyme on earth.  Rubisco is the most common enzyme in the world, too; every person on earth benefits from his or her own 12 to 25 pounds of these molecular machines, which process 15% of the total pool of atmospheric carbon per year.  For a long time, biochemists thought this enzyme was slow and inefficient.  That view is changing.  Rubisco now appears to be perfectly optimized for its job.
    Rubisco’s cute name is a handy anagram for the clumsier appellation ribulose bisphosphate carboxylase.  Tcherkez et al. first broke the paradigmatic logjam about this enzyme’s purported inefficiency with an article in PNAS,¹titled, “Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized.”  Howard Griffiths commented this week in Nature² about this paper and the new findings about its optimization.  Though his article referred to evolution seven times, and only mentioned design twice, the latter word seemed the most valuable player.
    There are four classes of Rubisco, some more efficient at fixing carbon than others.  Its reputation as a slow enzyme (2-8 catalytic events per second) may be unfair.  Carbon dioxide in gaseous form has to compete for access to the active site against the much more abundant and lighter oxygen.  Griffiths shows what a difficult job this molecule has to perform; no wonder it leaks somewhat.  But, as he explains, even the leaks are accommodated:

It is curious that Rubisco should fix CO₂ at all, as there is 25 times more O₂ than CO₂ in solution at 25°C, and a 500-fold difference between them in gaseous form.  Yet only 25% of reactions are oxygenase events at this temperature, and carbon intermediates ‘lost’ to the carbon fixation reactions by oxygenase action are metabolized and partly recovered by the so-called photorespiratory pathway.  Catalysis begins with activation of Rubisco by the enzyme Rubisco activase, when first CO₂ and then a magnesium ion bind to the active site.  The substrate, ribulose bisphosphate, then reacts with these to form an enediol intermediate, which engageswith either another CO₂ or an O₂ molecule, either of which must diffuse down a solvent channel to reach the active site.

This is a harder job than designing a funnel that will pass only tennis balls, when there are 500 times more ping-pong balls trying to get through.  Not only is Rubisco good at getting the best mileage from a sloppy process, it may actually turn the inefficiency to advantage.  Griffiths started by claiming, “evolution has made the best of a bad job,” but ended by saying that the enzyme’s reputation as “intransigent and inefficient” is a lie.  Why?  It now appears that “Rubisco is well adapted to substrate availability in contrasting habitats.”  This means its inefficiency is really disguised adaptability.
    Experimenters thought they could “improve” on Rubisco by mutating it.  They found that their slight alterations to the reactivity of the enediol intermediate drastically favored the less-desirable oxygenase reaction.  This only served to underscore the contortions the molecule must undergo to optimize the carboxylase reaction:

Such observations provided the key to the idea that in the active site the enediol must be contorted to allow CO₂ to attack more readily despite the availability of O₂ molecules.  The more the enediol mimics the carboxylate end-product, Tcherkez et al. conclude, the more difficult it is for the enzyme to free the intermediate from the active site when the reaction is completed.  When the specificity factor and selectivity for CO₂are high, the impact on associated kinetic properties is greatest: k_cat [i.e., the rate of enzyme catalytic events per second] becomes slower.
    So, rather than being inefficient, Rubisco has become highly tuned to match substrate availability.

Another finding about the inner workings of Rubisco bears on dating methods and climate models.  Scientists have known that Rubisco favors the lighter, faster-moving carbon isotope ¹²C over ¹³C.  By measuring the ratio of these stable isotopes in organic deposits, paleoclimatologists have inferred global carbon dioxide abundances and temperatures (knowing that Rubisco processes the isotopes differently).  That assumption may be dubious:

Several other correlates are also explained by this relationship.  For instance, Rubisco discriminates more against ¹³C than against ¹²C, the two naturally occurring stable isotopes in CO₂.  But when the specificity factor is high, the ¹³C reaction intermediate binds more tightly, and so carbon isotope discrimination is higher (that is, less ¹³C is incorporated); in consequence, the carbon-isotope signals used to reconstruct past climates should perhaps now be re-examined.  In contrast, higher ambient temperatures (30-40 °C) reduce the stability of the enediol, and Rubisco oxygenase activity and photorespiration rate increase.

Those considerations aside, Griffiths is most interested in two things: how this enzyme evolved, and whether we can improve on it.  If we can raise its carboxylation efficiency, we might be able to increase crop yields.  So far, genetic engineers have not succeeded.³
  As for the evolution of Rubisco, he mentions three oddball cases but fails to explain exactly how they became optimized for their particular circumstances – only that they are optimized.  Yet their abilities seem rather remarkable.  For instance, though the “least efficient” forms of Rubisco reside in microbes living in anaerobic sediments, where oxygen competition is not a problem, “One bacterium can express all three catalytically active forms (I, II and III), and switches between them depending on environmental conditions.”  In another real-world case, “some higher plants and photosynthetic microorganisms have developed mechanisms to suppress oxygenase activity: CO₂-concentrating mechanisms are induced either biophysically or biochemically.”  In another example, “Rubisco has not been characterized in the so-called CAM plants, which use a form of photosynthesis (crassulacean acid metabolism) adapted for arid conditions.”  These plants, including cacti and several unrelated species scattered throughout the plant kingdom, have other mechanisms for dealing with their extreme environments.  In every mention of evolution, therefore, Griffiths assumed it rather than explaining it: viz., “The systematic evolution of enzyme kinetic properties seems to have occurred in Rubisco from different organisms, suggesting that Rubisco is well adapted to substrate availability in contrasting habitats.”
    So, can we improve on it?  If so, given all the praise for what evolution accomplished, Griffiths seems oblivious to the implications of his own concluding sentence:

Other research avenues include manipulating the various components of Rubisco and cell-specific targeting of chimaeric Rubiscos.  Potential pitfalls here are that the modified Rubisco would not only have to be incorporated and assembled by crop plants, but any improved performance would have to be retained by the plants.  Finally, one suggestion is that we should engineer plants that can express two types of Rubisco – each with kinetic properties to take advantage of the degree of shading within a crop canopy.  Such rational design would not only offer practical opportunities for the future, but also finally give the lie to the idea that Rubisco is intransigent and inefficient.

What, students, is a synonym for “rational design”?

What Griffiths meant as a paper praising evolution is really a paper demonstrating intelligent design.  We dare any evolutionist to explain how this “highly-tuned” enzyme, with the optimized contortions of its intermediates and its “highly conserved” (i.e., unevolved) active site, arose by an unguided process, especially how a lowly bacterium – the simplest of organisms – evolved three forms of it and can switch between them depending on environmental conditions!  And don’t say it evolved because evolution is a fact.
   Here again, also, we see how further research is giving “the lie to the idea” that something in nature “is intransigent and inefficient.”  Evolutionists love to showcase examples of inefficiency in nature, to give the impression that any God or designer would not do such a bungled job.  The only bungling is in the theories of evolutionists who look at optimized, rational design in the face and can’t see a rational designer.  Human rational design applied to improving on nature’s engineering marvels does not support evolution, it supports intelligent design.  If human intelligence is required to copy or modify a design, one cannot say that the original design “emerged” by an unguided, purposeless, material process.  Why is that such a hard concept for the Darwinists to grasp?  Why can’t they see the illogic of their position?  As usual, they merely assume evolution can perform any engineering job necessary, even designing nanomachinery that exceeds our human capabilities.
   Notice the snippet about climate models in this story, also.  It goes to show that assumptions about the unobservable past, like foundations under a house of cards, can shift under new research.  Though Griffiths was not specific about the degree of alteration climate models might suffer, this is a point to remember whenever popular science reports glibly claim things like “218.24267 gazillion years ago, the atmosphere went through a period of global warming followed by a snowball earth.”
   You may never have heard about this indispensable enzyme that helps keep you breathing and gives you salad to eat (and, indirectly, meat from plant-eating animals).  Astrobiologists had better pay attention.  Mars and Venus have lots of carbon dioxide, but no Rubisco.  Earth has just enough CO2 to help moderate the atmospheric temperature, but not too much to cause a catastrophic greenhouse effect; that balance is maintained in part by this highly-tuned enzyme.  Our ability to read and write and think these thoughts owes to the convergence of numerous improbable factors, including our planet’s optimal distance from the sun, a global magnetic field, a planetary mass that retains the right ingredients but lets others escape, a transparent atmosphere, a star that produces radiation with just the right energy range for molecular reactions, and optimally engineered molecular machines in plants that can harvest that energy.  As a result, our lungs have air, our bodies have food, and our eyes have beauty and variety to enjoy.  If this looks like intelligent design, and if that has philosophical or religious implications, so be it.  Thank God for Rubisco.  

1) http://creationsafaris.com/crev200606.htm

¹Tcherkez et al., “Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized,” Proceedings of the National Academy of Sciences USA, published online before print April 26, 2006, 10.1073/pnas.0600605103 PNAS | May 9, 2006 | vol. 103 | no. 19 | 7246-7251.
²Howard Griffiths, “Plant biology: Designs on Rubisco,” Nature 441, 940-941 (22 June 2006) | doi:10.1038/441940a; Published online 21 June 2006.
³If and when they do, the benefit would be tuned for humans and their livestock, not necessarily for the ecology or atmosphere.

In spite of recent claims, the enzyme rubisco is optimally designed 3

http://www.reasons.org/articles/uprooting-a-%E2%80%9Cbad-design%E2%80%9D-argument

the complexity of rubisco has presented this research effort with difficulties. The protein consists of eight large and eight small subunits. The proper assembly of these subunits is difficult to achieve in the test tube. Likewise, genetically modified variants also face this problem, thus, hampering efforts to evaluate them for improved efficiency and selectivity.

A research team from Germany recently developed a means to effectively reconstitute rubisco in the laboratory.1 In a commentary on this work, biologist R. John Ellis takes the opportunity to deride rubisco, referring to it as a “superb example of unintelligent design.” Once again a challenge to the case for the Creator based on rubisco’s bad design.

work done in 2006, has changed the way biochemists view this enzyme. Rubisco’s slow turnover and struggles to discriminate between molecular oxygen and carbon dioxide stem from the featureless, nonpolar nature of both gases. In other words, rubisco’s confusion between oxygen and carbon dioxide is not due to a faulty design, but rather results from the inherent chemical nature of these two gases. To overcome this confusion, this enzyme slows down the carbon fixation reaction. Rubisco faces a trade-off between rate of reaction and discrimination between carbon dioxide and molecular oxygen. Rubisco is not poorly designed at all.

The biochemists that discovered this trade-off commented, “Despite appearing sluggish and confused, most Rubiscos may be near-optimally adapted to their different gaseous and thermal environments. If so, genetic manipulation can be expected to achieve only modest improvements in the efficiency of Rubisco and plant growth.”

In response to Ellis’ commentary, two agricultural scientists from Denmark maintain that the efficiency of rubisco has no bearing on crop yields, further undermining the view that this protein is poorly designed.5 They point out that increased crop production relates to larger leaves that capture more energy and prevent weeds from growing by creating more shade near the tree. They also note that crop physiologists have known for a long time that respiration and photorespiration do not detract from crop production. In fact, they assert that increasing the rate of photosynthesis—which would result if the efficiency of rubisco was increased—would reduce, not increase, crop yields. This counterintuitive effect stems from the fact that increased photosynthesis would consume a greater amount of energy because of higher respiration costs and higher demand for proteins to sustain the increased photosynthetic activity.

Rubisco no longer deserves its reputation as a poorly designed product of evolutionary processes, thus, uprooting another so-called example of a bad design.

Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized 2

http://www.pnas.org/content/103/19/7246.abstract

The cornerstone of autotrophy, the CO2-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates, an “abominably perplexing” puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO2/O2 specificity, in response to reducing atmospheric CO2:O2 ratios, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO2, O2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2/O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO2/O2 specificity and 12C/13C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature.



Light dependent activation of RuBISCO and Carbon dioxide fixation 1

Photosynthesis inplants can fall into three types C3 , C 4 and CAM.

C3 photosynthesis:
Photosynthesis is conversion of light energy into chemical energy utilizing chlorophyll (  LHC II.PS II and LHC I  PS I ) along with water and generating ATP, reducing NADP+ to NADPH + H+  and photolysis of water with release of Oxygen. 
 
Earlier Photosynthesis was divided into two type of reactions: Light reactions and dark reactions. 
It was assumed that Carbon fixation does not require light and the reaction is light independent. Here I would like to put together recent results from various research  groups  from USA, U.K.  Germany  France, and  Japan indicating that dark reaction also takes place in light only. It may not be possible to cite those who have contributed to this understanding but I acknowledge the work of all persons working in the field. 

Dark reaction requires presence of light and takes place at the same time as Light reaction: 
 In a way dark reaction is actually also light dependent Carbon fixation. Although  Calvin Cycle’ or ‘Carbon Reactions Pathway  do not Require Light energy to occur but they do require energy captured by light reactions and they occur at same time as light reactions.
This reaction takes place in light and is termed as “Light reaction”. C3 reduction  or Calvin cycle CO₂→ C3 → C6  (Melvin Calvin 1950s,  Nobel prize in 1961) was termed “dark reaction”.

 Enzymes&intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat analogous to the mitochondrial matrix. The Calvin Cycle, earlier designated  as the photosynthetic"dark reactions,"  is now called the carbon reactionspathway Carbon fixation. I propose to call it “light dependent carbon fixation” as this takes place during light only.    
  
 Light-activated e^- transfer is linked to pumping of H⁺ into thylakoid disks. This leads to increase of  pH in the stroma to about 8. This pH range ( 7.8 to pH 8.0 )is  required for activity of RuBisco to take place effectively.  Alkaline pH activates stromal Calvin Cycle enzymes RuBP Carboxylase, Fructose-1,6-Bisphosphatase and Sedoheptulose Bisphosphatase. 

The light-activated H⁺ shift is countered by Mg⁺⁺release from thylakoids to stroma. RuBP Carboxylase (in stroma) requires Mg⁺⁺ binding to carbamate at the active site.  Some plants synthesize a transition-state inhibitor, carboxyarabinitol-1-phosphate (CA1P), in the dark.RuBP Carboxylase Activase facilitates release of CA1P from RuBP Carboxylase, when it is activated under conditions of light by thioredoxin.  The activase is a member of the AAA family of ATPases, many of which have chaperone-like roles. 

Rubisco activity is dependent on light activation and dark inactivation: 
Regulation prevents the Calvin Cycle from being active in the dark, when itmight function in a futile cycle with Glycolysis & Pentose Phosphate Pathway, wasting ATP & NADPH. Light activates, or dark inhibits, the Calvin Cycle (previously called the “dark reaction”) in several ways.  Since photosynthetic light reactions produce ATP, the ATP dependence of RuBisCO activation provides a mechanism for light-dependent activation of the enzyme. 

Activation of RuBisco activase and coversion of inactive form of RuBisco  require electron transport and it can only take place in presence of light. Thioredoxin is a small protein with a disulfide that is reduced in chloroplasts via light-activated electron transfer. During illumination, the thioredoxin disulfide is reduced  to a dithiol by ferredoxin, a constituent of the photosynthetic light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase. Reduced thioredoxin activates several Calvin Cycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase Activase, by reducing disulfides in those enzymes to thiols.  Thus most of the important enzymes of Carbon dioxide fixation require light activation and hence Calvin cycle is Light dependent carbon fixation 

1) http://www.science20.com/humboldt_fellow_and_science/blog/light_dependent_activation_rubisco_and_carbon_dioxide_fixation-93130
http://crev.info/2006/06/rubisco_147highly_tuned148_for_fixing_atmospheric_carbon/
3) http://www.reasons.org/articles/uprooting-a-%E2%80%9Cbad-design%E2%80%9D-argument
2) http://www.pnas.org/content/103/19/7246.abstract

Enzymes in Plant Growth 1

Rubisco of higher plants belongs to the form I of the enzyme, found also in algae and in most photosynthetic bacteria. It is a complex protein, with eight large subunits (four large subunits) and eight small subunits  arranged in a L8S8 structure (four large subunit dimers along with eight non-catalytic small subunits capping the large ones).

The large subunits have the catalytic sites. Each subunit comprise an N-terminal domain and a larger C-terminal domain that forms a α /β -barrel; L2 dimers, formed by head-tail arrangement, have two active sites located at the L-L interface. The small subunits consist of four stranded antiparallel β-sheets with two α-helices; they are not essential for catalysis but provide structural stability to the Rubisco complex . L-subunits are synthesized from the single rbcL gene of the plastid genome; nucleus-encoded factors , chaperones  and post-translational modification of N-terminal domain  would help avoiding misfolding and protect the newly forming protein from proteolytic degradation.

Multiple copies of the rbcS gene, coding for the S-subunit, are located in the nucleus

Within the stroma, the S-subunits undergo further posttranslational modification (transit peptide cleavage, Met-1 aN- methylation) prior to assembly into L8S8 complexes.
The activity of Rubisco is highly regulated. The enzyme is inactive in the dark and is converted to an active form upon illumination. Activation is mediated by several environmental and systemic factors, including temperature, pH, light, heavy metal concentrations, natural inhibitors, and by the activity of an ancillary protein: Rubisco activase.

Prior to catalysis, Rubisco needs to be preactivated; activation is the result of the binding of CO2 to the 201-lysine residue near the catalytic site (position may slightly change depending on the species). The carbamate that is formed is then stabilized by Mg2+ binding. Carbamylation changes the conformation of the large subunit activating the enzyme that can bind RuBP and catalyzes a complex five-step reaction involving a CO2 and a water molecule before the release of two 3-phosphoglycerate (3PGA) molecules. Carbamylation is essential for Rubisco activation, as the non-carbamylated Rubisco binds RuBP too tightly to allow catalysis. The first, rate-limiting, step in carboxylation is the enolization of RuBP via the carbamate side chain; pH values lower than 8.0 may lead to the generation of Xylulose-1,5-bisphosphate that inhibit the enzyme activity.

Another protein, Rubisco activase, is also involved in mediating the light activation of Rubisco. This nucleus-encoded protein uses the energy of ATP to remove active-site bound sugar-phosphate inhibitors, such as 2 carboxyarabinitol 1-phosphate (CA1P) or xyulose-1,5- bisphosphate (XuBP), d-glycero-2,3-pentodiulose-1,5-bisphosphate (PDBP) and, under some conditions, RuBP itself. While XuBP and PDBP can be by-products of reaction intermediates, CA1P occurs naturally in the leaves of several plants and is a strong inhibitor of Rubisco. The affinity of Rubisco for CA1P is much stronger than that for RuBP, the substrate. As a result, CA1P, which accumulates in leaves during the night, inactivates Rubisco by blocking the binding sites. During the day (or on illumination), the bound CA1P is released from Rubisco by the concerted action of Rubisco activase and CA1P phosphatase.

The action of Rubisco activase may be crucial for maintaining Rubisco activity under low CO2 supply and the sensitivity of Rubisco activase to high temperature might explain the decrease in Rubisco efficiency under these environmental conditions. Besides the carboxylation reaction, Rubisco reacts with oxygen to form one molecule of 2-phosphoglycolate and one of PGA; this reaction is the first step of the photorespiration pathway that leads to the release of previously fixed CO2, NH3 and energy. Due to photorespiration, C-fixing reaction has a reduced efficiency and a large amount of protein is needed to support adequate photosynthetic rates (Rubisco accounts for an average of 50% of leaf protein). Photorespiration if favoured by prolonged drought stress conditions and high temperature.

1) http://www.esciencecentral.org/ebooks/enzymes/enzymes-in-plant-growth.php
2) http://www.nature.com/nrm/journal/v14/n12/fig_tab/nrm3702_F5.html

http://subversify.com/2011/12/23/rubisco-and-evolution/

it’s a reasonable guess that the Rubisco was one of the very first enzymes created by the earliest organisms on our planet.

http://jxb.oxfordjournals.org/content/59/7/1555.full

Despite apparent differences in amino acid sequence and function (in the case of the RLPs), the secondary structure of the large (catalytic) subunit is extremely well conserved throughout different forms of Rubisco


http://plantsinaction.science.uq.edu.au/edition1/?q=content/2-1-4-properties-rubisco

Something like 1000 million years of evolution has still not resulted in a ‘better’ Rubisco. Such a highly conserved catalytic protein is an outcome of thermodynamic and mechanistic difficulties inherent to this reaction. Rubisco first evolved ( sic ) when the earth’s atmosphere was rich in CO2, but virtually devoid of O2.

Photorespiration Found Not Wasteful

http://farmprogress.com/story-photorespiration-found-not-wasteful-9-2089

A biological process in plants, thought to be useless and even wasteful, has significant benefits and should not be engineered out -- particularly in the face of looming climate change, says a team of UC Davis researchers.

The researchers have found that the process, photorespiration, is necessary for healthy plant growth and if impaired could inhibit plant growth, particularly as atmospheric carbon dioxide rises as it is globally. Their findings are published in the Proceedings of the National Academy of Sciences.

A short history of RubisCO: the rise and fall (?) of Nature’s predominant CO2 fixing enzyme

https://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S095816691730099X

Although RubisCO has been intensively studied, its evolutionary origins and rise as Nature’s most dominant carbon dioxide (CO2)-fixing enzyme still remain in the dark.

Even though evolution is generally considered to be a
highly creative force, its nature is actually rather conservative. Once a biological solution is found by evolution,
the space to explore new evolutionary paths dramatically narrows, as evolution rather tends to work in a tinkering
fashion by improving and recombining existing parts and
pieces.


My comment:
Creativity is always attributed to intelligent action
Solution finding is a goal-oriented process depending on intelligence.
Exploring spaces is also something that only intelligence does.
improving and recombining is a goal-oriented process, and as well depending on intelligent action.

It is remarkable, how teleological language is used in evolutionary papers. Shamelessly. And the authors know, the majority of the readers are blind to the fact that evolution is not goal-oriented, nor conscious to be able to take all those actions.

Rubisco 's amazing evidence of design
https://reasonandscience.catsboard.com/t1554-the-rubisco-enzymes-amazing-evidence-of-design

Biophysical analysis of the structural evolution of substrate specificity in RuBisCO
https://pubmed.ncbi.nlm.nih.gov/30790657/