Where is rubisco

Figure 2. Model summarizing the roles of different chaperones in Rubisco assembly. After import into chloroplast and cleavage of its transit peptide, RbcS S folds spontaneously, or with the help of a chaperone.

RbcS subunits could either displace the chaperones in a final chaperone-RbcL intermediate to form the holoenzyme L 8 S 8 , or interact with chaperones and RbcL in earlier stages of the assembly.

Continuous and dashed arrows indicate certain and speculative nature of each step, respectively. In the most recent model, however, sequential functions have been proposed, during which Raf1 and RbcX are involved in the earlier RbcL oligomerization steps, and their replacement by Bsd2 mediates a later stabilization step of the RbcL 8 core.

According to this model, RbcS may only have to replace Bsd2 before formation of the holoenzyme Aigner et al. Many question marks surround this model. What is the precise role of Raf2? Is RbcS folded spontaneously or in need of chaperone assistance to reach conformation compatible for RbcL binding? Do RbcX and Raf1 act in parallel or cooperatively? How RbcS displaces Bsd2? Are there additional factors involved in Rubisco biogenesis?

Revealing the sequential steps of assembly, as well as the precise role of different chaperone paralogs is the next challenge. Further in vitro and in vivo experiments seem essential in unraveling the assembly steps and characterizing the unique structural and functional properties of the different factors. Reconstitution of Arabidopsis Rubisco in vitro was previously attempted. The results showed that RbcL subunits stayed bound to chaperonins and did not assemble into any type of oligomers or holoenzyme despite the presence of all assembly factors except Bsd2 Hauser, , as one would expect in light of the recent work.

Whether the entire cohort of assembly factors, their exact levels, and an accurate timing of theirs functions, would be sufficient for in vitro assembly, is yet to be determined. Evolution has invested tremendous resources in the fine-tuning of various folding and assembly factors and their compatibility with RbcL and RbcS in chloroplast. Further genetic and biochemical studies are necessary for complete, in detail understanding of this complex pathway.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. 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 thank Dr. Celeste Weiss and Prof. David Stern for critically reading the manuscript.

Department of Agriculture, under award number Aigner, H. Plant RuBisCo assembly in E. Science , — Andersson, I. Structure and function of Rubisco. Plant Physiol. Badger, M. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO 2 acquisition by the CBB cycle. Bai, C. Protomer roles in chloroplast chaperonin assembly and function.

Plant 8, — Baneyx, F. Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin Barraclough, R.

Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bishopshate carboxylase in isolated intact pea chloroplasts.

BBA Sect. Nucleic Acids Protein Synth. Belcher, S. Large-scale genetic analysis of chloroplast biogenesis in maize. Acta Bioenerg. Bertsch, U. PubMed Abstract Google Scholar. Bloom, M. Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase. Cell Biol. Bonshtien, A. Differential effects of co-chaperonin homologs on cpn60 oligomers. Cell Stress Chaperones 14, — Significance of the N-terminal domain for the function of chloroplast cpn20 chaperonin.

Bracher, A. Crystal structure of a chaperone-bound assembly intermediate of form I Rubisco. Biogenesis and metabolic maintenance of Rubisco. Plant Biol. Brutnell, T. Plant Cell 11, — Cloney, L. Assessment of plant chaperonin gene function in Escherichia coli. Expression of plant chaperonin genes in Escherichia coli. Dereeper, A. BMC Evol. Nucleic Acids Res. Dickson, R.

Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding. Dobberstein, B. In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhardtii. Opposing effects of folding and assembly chaperones on evolvability of Rubisco.

Ellis, R. Molecular chaperones: the plant connection. Emlyn-Jones, D. Plant Cell Physiol. Endrizzi, J. Crystal structure of DCoH, a bifunctional, protein-binding transcriptional coactivator. Erb, T. A short history of RubisCO: the rise and fall? Feiz, L. A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants. Plant J. Plant Cell 24, — Feller, U. Rubiscolytics: fate of rubisco after its enzymatic function in a cell is terminated.

Friso, G. 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. Inorganic phosphate binds to three sites in the structure from tobacco Rubisco Duff et al. Two of the sites are within the active site and coincide with the positions occupied by the P1 and P5 phosphate groups of RuBP. Because of the shift of the position of the phosphates when the active site closes, these sites consist of two distinct subsites; a distal and a proximal site for the P1 group, and a lower and an upper site for the P5 group in the nomenclature of Duff et al.

Closing of the active site causes a shift of the phosphate groups of the substrate between these sites and shortening of the inter-phosphate distance resulting in tight binding of the ligand Duff et al. The third phosphate ion binds at a surface site where it takes the place occupied by the side chain of the carboxy-terminal Asp in the closed complexes.

The binding of inorganic phosphate to the two types of sites, the latch site and the P5 phosphate site, has been investigated by mutagenesis in Rubisco from Synechocystis sp. It was found that mutations at both sites abolished the phosphate-stimulated activation, whereas mutation of the P5 site interfered with the inhibition of Rubisco catalysis by phosphate.

Although several questions remain to be answered, it may be possible to interpret the dual role of phosphate ions and phosphorylated compounds as activators of carbamylation and as competitive inhibitors to the sugar substrate by invoking two types of binding sites with different affinities for the effector molecules Duff et al. When binding at the two sites within the active site, these compounds interfere with substrate binding and behave as competitive inhibitors.

When binding at the latch site, the effectors compete with the closing mechanism and again act as inhibitors. However, at the same time they stabilize the carbamylated enzyme in a manner that appears similar to how Rubisco activase works. One might speculate that the binding of effectors at the latch site destabilizes the closed inactivated complex with RuBP and helps the enzyme to open, rid itself of the ligand, and reactivate.

If this were the case, binding at the latch site would stabilize the carbamylated form of the enzyme. Also, binding of these compounds at the two types of sites with different affinities might explain the biphasic kinetics. Rubisco activase is intimately involved in the light-dependent regulation of Rubisco by relieving the inhibition of phosphorylated compounds that bind to both carbamylated and non-carbamylated Rubisco active sites Portis, ; Portis et al.

The three-dimensional structure of activase is not known, nevertheless, evidence is slowly gathering towards its function. Members of the family typically form large ring-structures and interact with other proteins, in a way characteristic for molecular chaperones.

Site-directed mutagenesis studies of Rubisco from C. Instead, the mutant enzymes now could be activated by the tobacco enzyme. Thus the binding of activase at this site is likely to interfere with the closing mechanism. Portis has presented a mechanism in which activase interacts with Rubisco and opens the active site by a reversal of the closing mechanism discussed above. Binding of activase at or around residue 89 on Rubisco initiates movement of the amino-terminal domain towards the open conformation leading to the opening of loop 6 and the carboxy-terminus.

Residues in the carboxy-terminal region of activase and have been identified that may directly interact with residues on the loop in Rubisco Li et al. It is a sobering thought that our life depends on an enzyme that is so incomplete. Rubisco, or rather the Rubisco catalytic large subunit, emerges as a molecule in need of its helpers.

It is not yet known what additional chloroplast factors are needed for proper assembly in plants, but it is reasonable to assume that some additional helper protein, similar to the RbcX assembly chaperone found in cyanobacteria discussed above , is needed in order to form the large subunit L8 core.

In analogy with what is known about the assembly process in cyanobacteria Saschenbrecker et al. Once a functional enzyme has been assembled, catalysis carboxylation or oxygenation depends on proper activation by formation of a lysyl-carbamate that also plays a crucial role during several stages of catalysis. The activation process requires the help of Rubisco activase and several effector molecules. Information on the molecular level are still lacking, but as discussed above, several investigations point to a role for the carboxy-terminus of the large subunit in several of these processes.

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Volume Article Contents Abstract. Structure overview. Synthesis and assembly. Concluding remarks. Catalysis and regulation in Rubisco. Oxford Academic. Revision received:. Select Format Select format. Permissions Icon Permissions. Open in new tab Download slide. Google Scholar Crossref. Search ADS. High speed high resolution data collection on spinach Rubisco using a Weissenberg camera at the Photon Factory.

Crystal structure of the active site of ribulose-bisphosphate carboxylase. A theoretical-study of the singlet-triplet energy-gap dependence upon rotation and pyramidalization for 1,2-dihydroxyethylene: a simple-model to study the enediol moiety in rubisco substrate. Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits.

Google Scholar PubMed. The biochemistry of plants: a comprehensive treatise, Vol. Regulation of ribulose 1,5-bisphosphate carboxylase oxygenase activation by inorganic-phosphate through stimulating the binding of the activator CO 2 to the activation sites. Interaction of sugar phosphates with the catalytic site of ribulose-1,5-bisphosphate carboxylase.

Protein-synthesis in chloroplasts. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea-chloroplasts. Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids.

The transition between the open and closed states of Rubisco is triggered by the inter-phosphate distance of the bound bisphosphate.

A kinetic characterization of slow inactivation of ribulosebisphosphate carboxylase during catalysis. Slow inactivation of ribulose bisphosphate carboxylase during catalysis is not due to decarbamylation of the catalytic site. Slow inactivation of ribulose bisphosphate carboxylase during catalysis is caused by accumulation of a slow, tight-binding inhibitor at the catalytic site. 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. In fact, photosynthetic bacteria build a smaller rubisco shown on the right, taken from PDB entry 9rub composed of only two chains, which performs its catalytic task just as well. So, why do plants build a large complex? The answer might lie in the crowded conditions under which rubisco performs its job. By packing many chains together into a tight complex, the protein reduces the surface that must be wetted by the surrounding water.

It is mandatory to procure user consent prior to running these cookies on your website. Twitter Linkedin. Jun 29, A key enzyme in green leaves: the most abundant protein present on earth But, what is RuBisCo? Nutritional Value RuBisCO is of potential interest for human consumption due to its high nutritional value. Post Views: 15, Categories Uncategorized 6.

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