How many substrates of rubisco are there

Indeed, although published comments Griffiths, ; Gutteridge and Pierce, on the paper of Tcherkez et al. However, a method to reduce the sequence-search space for RuBisCO has since been reported in a patent Gready and Kannappan, In summary, there is still wide conjecture in the literature regarding the mechanisms by which plants ultimately regulate photosynthesis Igamberdiev, , and the absolute limitations of RuBisCO functionality have only been partly explored, as recent studies Hanson, ; Young et al.

Consequently, the potential for increasing both the catalytic turnover and relative specificity in higher plants with the view to improving photosynthesis remains to be fully tested. As argued Hanson, , kinetic data for a wider diversity of RuBisCOs are much needed and will likely prove useful in guiding the reengineering of higher-plant RuBisCOs with both significantly higher turnover rate and specificity.

Our analysis suggests that such simultaneous improvement in both specificity and turnover rate is possible, and that competing selection pressures of activity and stability better explain the nature of constraints. Improved understanding of these competing selection pressures is much needed. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We sincerely thank Dr. Andrey Bliznyuk for checking the manuscript and helpful comments. We thank the reviewers for helpful comments. Andersson, I. Catalysis and regulation in Rubisco. Badger, M. Dordrecht: Springer Netherlands , — PubMed Abstract Google Scholar.

The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO 2 -concentrating mechanisms in algae. J, Bot. Bainbridge, G. Engineering Rubisco to change its catalytic properties. Bar-Even, A. The moderately efficient enzyme: futile encounters and enzyme floppiness.

Biochemistry 54, — The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50, — Carmo-Silva, A. Rubisco activities, properties, and regulation in three different C 4 grasses under drought. Carmo-Silva, E. Optimizing Rubisco and its regulation for greater resource use efficiency. Plant Cell Environ. Cleland, W. Mechanism of Rubisco: the carbamate as general base.

Farquhar, G. Models describing the kinetics of ribulose biphosphate carboxylase-oxygenase. Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends. Ghannoum, O. Plant Physiol. Gready, J. Process for Generation of Protein and Uses Thereof. Washington, DC: U. Patent and Trademark Office.

Griffiths, H. Plant biology: designs on Rubisco. Nature , — Gutteridge, S. A unified theory for the basis of the limitations of the primary reaction of photosynthetic CO 2 fixation: was Dr. Pangloss right? Hanson, D. Breaking the rules of Rubisco catalysis. Igamberdiev, A. Control of Rubisco function via homeostatic equilibration of CO 2 supply. Plant Sci. Jordan, D. Kannappan, B.

Redefinition of Rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues. Koralov, L. Theory of Probability and Random Processes. Berlin; Heidelberg: Springer-Verlag. Google Scholar. Kubien, D. The biochemistry of Rubisco in Flaveria. Lorimer, G. The carboxylation and oxygenation of ribulose 1,5-bisphosphate: the primary events in photosynthesis and photorespiration.

McNevin, D. Determining RuBisCO activation kinetics and other rate and equilibrium constants by simultaneous multiple non-linear regression of a kinetic model. Parry, M. Rubisco activity and regulation as targets for crop improvement. Peterhansel, C. Metabolic engineering towards the enhancement of photosynthesis. Pierce, J. The crystal structure of the RbcX monomer is a four-helix bundle. Formation of a homodimer of the 15 kDa protein creates a central peptide-binding grove that recognizes a conserved and partially unfolded carboxy-terminal peptide of the large subunit.

Based on their studies, Saschenbrecker et al. This process, in turn, is facilitated by the dynamic assembly between RbcX and the core complex RbcL8. The degree of sequence conservation in cyanobacterial RbcX homologues suggest they have similar architecture and this was confirmed by crystal structures of RbcX from Anabaena sp. RbcX homologues identified in plants are more distant, but share the register and structural residues. In the crystal structure of the DE mutant of Rubisco from Chlamydomonas reinhardtii Satagopan and Spreitzer, the carboxy-terminus is disordered in seven of the eight large subunits Karkehabadi et al.

However, in one large subunit, it is stabilized by a crystal contact in a way that may mimic the interaction with a chaperone. Thus the RbcL carboxy-terminus appears to have multiple functions; it participates in conformational transitions during catalysis and also in forming and stabilizing the RbcL8 core complex during assembly.

The role of RbcX appears to be to protect this sequence from incorrect interactions during these processes. The carbamylated Lys 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, and hydration of the resulting ketone, carbon—carbon scission, and stereospecific protonation of the resulting carboxylate of one of the product 3PGA.

Several, if not all, of these steps involve acid—base chemistry. Considerable time and effort has been spent to identify enzyme residues that participate in catalysis. High-resolution crystal structures have provided steric constraints, while chemical modification, site-directed mutagenesis, molecular dynamics calculations, and quantum chemical analyses have added mechanistic and energetic constraints reviewed in Andrews and Lorimer, ; Hartman and Harpel, ; Cleland et al.

Here the main points are recapitulated as far as they are known. The main reactions catalysed by Rubisco, carboxylation and oxgenation of RuBP. This requires removal of the proton at C3 and protonation of the carbonyl group at C2.

Deprotonation by an enzyme base is a typical feature of enzymes that catalyse enolization reactions Knowles, and, in the case of Rubisco, the nature of the base has been intensely debated Hartman and Harpel, ; Cleland et al.

The steric constraints imposed by the crystal structures singled out the non-Mg-co-ordinated carbamate oxygen on K as the most likely candidate for the base Andersson et al.

This may now seem trivial, but required X-ray data to high resolution that could only be obtained with the introduction of larger and better detectors Andersson et al. The involvement of K would be difficult to prove or disprove by mutagenesis, because mutation of K would render the enzyme inactive. Computational methods have been used to probe the energetics of the reaction either using minimal 3-carbon or 5-carbon transition structures in vacuo Tapia et al.

These calculations stress the importance carbamylated Lys in the enolization. Calculations by King et al. Lys may thus correspond to the essential acid implicated in measurements of the pH profile in the deuterium isotope effect Van Dyk and Schloss, Deprotonation of C3 by Lys and protonation of O2 by Lys is in accordance with the crystal structures that place Lys on the opposite side of the transition state analogue with respect to the carbamylated Lys Knight et al.

Presumably, these steps help to direct the gaseous substrate to the C2 atom, otherwise C3 would be as likely to react. The moulding of the C2 and C3 centres in a cis out-of-plane conformation around the C2—C3 bond in the transition structure provides the necessary activation for the reaction to proceed Andres et al.

In the next step, CO 2 or O 2 compete for the enediolate. The competing reaction of O 2 with RuBP appears to be an inevitable consequence of the mechanism of carboxylation Andrews and Lorimer, ; Cleland et al. The oxygenation reaction is the first step in photorespiration, a process that salvages some of the carbon of 2-phosphoglycolate at the cost of energy and evolved CO 2 Andrews and Lorimer, The oxygenase reaction is an intrinsic characteristic of Rubisco, the extent of which depends on the properties of the particular enzyme studied.

Thus, the key to the efficiency of any particular Rubisco enzyme lies hidden in the fine details of its three-dimensional structure and this has motivated intense research with the ultimate aim to improve crop yields reviewed in Spreitzer and Salvucci, ; Parry et al. Thus the relative rates for carboxylation and oxygenation are defined by the product of the specificity factor and the ratio of CO 2 to O 2 concentrations at the active site.

The specificity values of Rubisco enzymes from different origins differ substantially Jordan and Ogren, Some photosynthesizing bacteria have the lowest specificity values 5—40 , red algae have the highest — , whereas higher plants and green algae have intermediate specificity values in the range of 60— However, it appears that positive selection towards higher specificity has occurred at the cost of overall carboxylation rate, because an inverse correlation between specificity and turnover rate V c or k cat for carboxylation has been observed Jordan and Ogren, ; Bainbridge et al.

Some organisms have evolved mechanisms carboxysomes, pyrenoids, C 4 - and CAM metabolisms that concentrate CO 2 at the carboxylation site Dodd et al. Thus the specificity factor is but one parameter that determines the net efficiency of Rubisco enzymes, but it can serve as an important first diagnostic parameter to indicate changes in efficiency of engineered Rubisco enzymes. McNevin et al. One of the key players in the reaction catalysed by Rubisco is the magnesium ion.

Apart from the carbamylated Lys which provides a monodentate ligand, the magnesium ion is liganded by two monodentate carboxylate ligands provided by Asp and Glu Fig. 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 Taylor and Andersson, a and this presumably also includes residues outside the immediate co-ordination sphere.

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.

Residues implicated in the reaction mechanism are highlighted. The crystal structures show Lys positioned to facilitate the addition of the gaseous substrate Fig. During catalysis, Rubisco undergoes a conformational change, which serves to close the active site and prevent access of water during the reaction.

Rubisco structures can be divided into two states Duff et al. Packing of the carboxy-terminal strand residues to the carboxy-terminal end against loop 6 completes the closure Schreuder et al. Two strictly conserved glycine residues, Gly and Gly, maintain flexibility in the hinge of loop 6.

The other strictly conserved residue, Lys, is located at the tip of the loop and interacts with the incoming gaseous substrate during catalysis. The Lys 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 CO 2 Knight et al.

The exact timing of the closure of the active site is not known. The inhibitor 2CABP forms a tight-binding closed complex ideal for crystallization. Complexes with ligands that bind less tightly, such as the natural substrate RuBP or the product 3-PGA have been more difficult to crystallize Lundqvist and Schneider, , ; Taylor et al. The resulting structure is open, with loop 6 partially retracted Taylor et al.

It appears that the presence of RuBP only is not enough to trigger the closing of the active site. Also, soaking 2CABP into crystals of calcium-activated Rubisco induces closing of the active site in the crystal Karkehabadi et al. This indicates that it is not the calcium ion per se that prevents the conformational switch. It may be that the interaction of the substrate—CO 2 with Lys is required for the shortening of the interphosphate distance of the substrate Duff et al.

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

Similar results were obtained in Synechococcus Rubisco Gutteridge et al. Substitution of the Val side chain by the smaller Ala weakened these interactions considerably and created a small cavity that was partly filled by solvent. Val and Thr flank Lys located at the apex of loop 6, and it seems likely that the mutation could destabilize the loop or alter its flexibility in a way that could influence the catalytic properties of the mutant enzymes.

Similar conclusions were drawn from the observation of altered catalysis in the tobacco enzyme induced by a LV mutation, which caused a reduction in specificity and altered sensitivity to inhibitors Whitney et al. This displacement also brings Thr closer to Ala It thus appears that specificity is restored by two fundamentally different mechanisms in the two revertant enzymes.

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 Schreuder et al. As shown by site-directed mutagenesis, the carboxy-terminus is not absolutely required for catalysis, but is needed for maximal activity and stability Morell et al. Residue Asp 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 Duff et al.

Directed mutagenesis and chloroplast transformation in C. The crystal structure of DE Karkehabadi et al. It appears that the mutations disrupt contacts of residue with Arg and His This may cause a destabilization of the underlying loop 6 with consequences for catalysis and specificity.

Hydration at C3 may occur either simultaneously with carboxylation, or as a separate step. A similar situation is observed in the enzyme from Rhodospirillum rubrum Lundqvist and Schneider, No further details are known for this step, but it is noted that the carbamate group of Lys and the side chain of His are within hydrogen bonding distance to the water molecule in the spinach structure and thus well placed to assist the hydration step.

It is possible that key active site residues, such as the carbamylated Lys, may assist several of the partial reactions. One example is the final stereospecific protonation of the carboxylate ion of upper 3PGA. Structurally, protonated Lys is ideally positioned to donate this proton Taylor and Andersson, a and experimental evidence indicates that deletion of the side chain interferes with this step Harpel and Hartman, If Lys assists in the delivery of a proton to C2 in the enolization reaction as suggested King et al.

Such proton relocations could presumably occur as internal protonation steps within the active site. The enzyme is also subject to control by the redox status of the chloroplast Moreno et al. In addition, a number of other metabolites mainly phosphorylated compounds also present in the chloroplast may interfere or interact with the activating process reviewed in Parry et al. The interaction of Rubisco with anions and phosphorylated sugar compounds is complex.

It has been estimated that a large fraction of the phosphorylated sugar compounds in the chloroplast is bound to Rubisco Pettersson and Ryde Pettersson, Modulation of the activity of Rubisco has been observed by the binding of various effectors such as anions, sulphate, inorganic phosphate or phosphorylated sugars such as pyridoxal 5-phosphate, phosphogluconate, and NADPH.

At limiting concentrations of CO 2 , effectors may act positively or negatively by either promoting or inhibiting activation Whitman et al. Some of these effectors, but not all, act as competitive inhibitors of the enzyme with respect to the substrate RuBP. As a further complication, some of the effectors give rise to biphasic kinetics, suggesting negative co-operativity between active sites Whitman et al. The purified enzyme from higher plants becomes progressively inhibited during in vitro catalysis by strongly inhibitory phosphorylated compounds that bind in the active site, a process termed fallover Edmondson et al.

Some compounds, notably xylulose-1,5-bisphosphate XuBP , are formed by the incorrect stereochemical reprotonation of the enediolate bound at the catalytic site and often bind to the non-carbamylated enzyme with high affinity. This leads to a tight complex that lacks the metal ion and is catalytically inactive McCurry and Tolbert, ; Zhu and Jensen, ; Newman and Gutteridge, ; Taylor et al.

An inactive complex with the natural substrate, RuBP, appears to play an important role in the light-dependent regulation of Rubisco activity in vivo. As part of this mechanism RuBP binding to decarbamylated enzyme locks the enzyme in an inactivated form Jordan and Chollet, CA1P resembles 3-keto-2CABP and accumulates in plants in the dark and in low-light conditions, often to amounts exceeding Rubisco active site concentrations 5 mM. It has an affinity for the activated form of the enzyme in the nanomolar range Gutteridge et al.

In binding to the carbamylated enzyme, 2CA1P effectively prevents its interaction with RuBP and inactivates the enzyme. Release of CA1P from the active site is facilitated by Rubisco activase Robinson and Portis, followed by degradation by a specific phosphatase Gutteridge and Julien, ; Holbrook et al. The level to which CA1P can accumulate varies considerably in plant species. Phaseolus vulgaris has a very large capacity for CA1P accumulation whereas in wheat, Arabidopsis, and spinach low levels of accumulation are observed Moore et al.

It may be that species that show low accumulation of CA1P primarily use changes in the extent of carbamylation and binding of decarbamylated enzyme to RuBP as a means for light regulation.

The biphasic kinetics can be interpreted in more than one way. Biphasic inhibition may be caused by isomerization of a loose and reversible complex to a much tighter complex Edmondsson et al. Negative co-operativity is difficult to prove or disprove and the behaviour can often be mimicked in a sample contaminated by a less active or slightly denatured form of the protein, or it can be linked to ageing processes in the protein.

The kinetic behaviour may also be influenced by simultaneous binding to an effector site outside the active site. The first crystal structures gave no information on such a site Schneider et al. Therefore, an important clue was the observation of the binding of inorganic sulphate or phosphate at a surface site in the structure of non-carbamylated Rubisco from tobacco Curmi et al. Because of its location at a latch site that holds down the carboxy-terminus in the closed complexes, it could not be detected in the ligand-bound complexes Knight et al.

In spite of its central role, rubisco is remarkably inefficient. As enzymes go, it is painfully slow. Typical enzymes can process a thousand molecules per second, but rubisco fixes only about three carbon dioxide molecules per second.

Plant cells compensate for this slow rate by building lots of the enzyme. Chloroplasts are filled with rubisco, which comprises half of the protein. This makes rubisco the most plentiful single enzyme on the Earth. Rubisco also shows an embarrassing lack of specificity.

Unfortunately, oxygen molecules and carbon dioxide molecules are similar in shape and chemical properties. In proteins that bind oxygen, like myoglobin, carbon dioxide is easily excluded because carbon dioxide is slightly larger. But in rubisco, an oxygen molecule can bind comfortably in the site designed to bind to carbon dioxide.

Rubisco then attaches the oxygen to the sugar chain, forming a faulty oxygenated product. The plant cell must then perform a costly series of salvage reactions to correct the mistake. Rubisco from spinach left and photosynthetic bacteria right. Plants and algae build a large, complex form of rubisco shown on the left , composed of eight copies of a large protein chain shown in orange and yellow and eight copies of a smaller chain shown in blue and purple.

The protein shown here is taken from spinach leaves coordinates may be found in the PDB entry 1rcx ; the tobacco enzyme may be found in 1rlc. Many enzymes form similar symmetrical complexes. Often, the interactions between the different chains are used to regulate the activity of the enzyme in the process known as allostery. Rubisco, however, seems to be rigid as a rock, with each of the active sites acting independently of one another.