Cited By. This article is cited by 37 publications. Peter L. Cummins, Jill E. The Journal of Physical Chemistry B , 15 , The Journal of Physical Chemistry B , 13 , Cummins, Babu Kannappan, Jill E. The Journal of Physical Chemistry B , 12 , Biochemistry , 52 5 , Journal of the American Chemical Society , 7 , Fedunov , Victor A. International Journal of Molecular Sciences , 22 18 , Catalysts , 11 7 , Journal of Molecular Evolution , 89 , Castro , Millena P.
Ferreira , Caterina G. Marques Netto. Metalloenzyme mechanisms correlated to their turnover number and metal lability. Current Research in Chemical Biology , 1 , Farquhar , Michelle L. Coote , George H. Lorimer , Guillaume Tcherkez. Proceedings of the National Academy of Sciences , 39 , Bennie , Kara E.
Ranaghan , Adrian J. Journal of Computational Chemistry , 41 24 , Danielache , Shinkoh Nanbu. Theoretical analysis of the kinetic isotope effect on carboxylation in RubisCO. Journal of Computational Chemistry , 41 11 , Electronic structure benchmark calculations of inorganic and biochemical carboxylation reactions. Journal of Computational Chemistry , 40 13 , Risso , Jose M. Sanchez-Ruiz , Spencer M. Whitney , Miguel Alcalde. The small subunit is not essential for catalysis, because the large subunit octamer still retains some carboxylase activity Andrews, ; Gutteridge, ; Lee et al.
A further distinction of the form I enzymes can be made Delwiche and Palmer, ; Tabita, between green-type enzymes forms IA and B from cyanobacteria, eukaryotic algae, and higher plants and red-type enzymes forms IC and D from non-green algae and phototropic bacteria. The most common form of Rubisco from plants, cyanobacteria, and green algae is a hexadecamer of eight large and eight small subunits.
A The L8S8 hexadecamer from spinach viewed along the 2-fold symmetry axis. Large subunits are blue, small subunits are yellow, and the substrate mimic 2CABP is displayed as green spheres.
The fold of one L2S2 unit is highlighted. B View of the hexadecamer along the 4-fold axis. C The L2S2 unit in A with mobile elements amino-terminal loop, loop 6, carboxy-terminus highlighted in red. The form II enzyme is a dimer of large subunits L2 n and lacks small subunits. The form II enzyme was initially discovered in purple non-sulphur bacteria, but has also been found in several chemoautotropic bacteria reviewed in Shively et al.
Several non-sulphur phototropic bacteria, i. Rhodobacter sphaeroides , R. The first crystal structure of Rubisco was from the dimeric form II enzyme from Rhodospirillum rubrum Schneider et al. Rbc L sequences have also been identified in archaea and assigned to a separate group, form III. With respect to quaternary structure, the archaea are diverse and comprise L2, L8, and L10 enzymes.
Rubisco from Thermococcus kodakaraensis Kitano et al. The crystal structure of Rubisco from Pyrococcus horikoshii consists of an octamer of large subunits, L8 PDB codes 2cxe, 2cwx, 2d Putative Rubisco sequences differing from form I and II sequences have been identified in organisms that do not use CO 2 as the major source for carbon Tabita, 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 reviewed in Andersson and Taylor, ; Andersson and Backlund, Despite this relatively large divergence on the level of sequence, differences are localized to a few loops Andersson and Backlund, The active site is located at the intra-dimer interface between the carboxy-terminal domain of one large subunit and the amino-terminal domain of the second large subunit in the L2 dimer.
In the hexadecameric molecule, the dimers are arranged such that the eight active sites face the outside solvent Fig. Two loop regions in the amino-terminal domain of the second large subunit in the dimer contribute additional residues to the active site. Thus, the functional unit of Rubisco is an L2 dimer of large subunits containing two active sites. The small subunit is more diverse. The length of the loop is variable, the shortest occurs in Rubisco from prokaryotes and non-green algae, and the longest in green algal Rubisco Karkehabadi et al.
The function of the small subunit is enigmatic. Its structural arrangement, covering a substantial area at two opposite ends of the L-subunit octamer makes it reasonable to assume a structural chaperone-like function of the small subunit. In this capacity it influences both holoenzyme stability and catalytic performance Spreitzer et al.
The Rubisco large subunit is encoded by a single gene in the chloroplast genome and is synthesized by the plastid ribosome. In plants, the small subunit is coded by a family of closely related nuclear genes and synthesized in the cytosol reviewed in Spreitzer, The synthesis and assembly of the Rubisco holoenzyme, involving the co-ordinated control of chloroplastic and cytosolic processes, have been shown to require the assistance of ancillary proteins termed molecular chaperones Barraclough and Ellis, ; reviewed in Gatenby and Ellis, The plastid chaperones belong to a large family named chaperonins Hemmingsen et al.
Two major types are encountered, the chaperonin 60 cpn60 and chaperonin 10 cpn Despite the close homology between the bacterial and plastid chaperonins Hemmingsen et al. Studies on the structure and function of a cyanobacterial protein, RbcX, point to a role for RbcX as an assembly chaperone Saschenbrecker et al.
RbcX has been shown to promote the synthesis and assembly of active recombinant cyanobacterial holoenzyme in E. This is the case for instance in Anabaena sp. PCC Tanaka et al. Saschenbrecker et al. 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. Step 5. The C3-C2 bond is cleaved forming one of the 3-phosphoglycerate products and an enolate of another 3PG molecule.
Step 6. The enolate intermediate tautomerizes into 3PG, this is assisted by protonation from Lys and the Lys carbamate. Ribulose-bisphosphate carboxylase type II. Reference Protein and Structure. Sequence P 4. Click To Show Structure. Show Active Center.
Enzyme Reaction EC: 4. CHEBI: Download: Image , Marvin File. Catalytic Residues Roles Residue Roles LysA electrostatic stabiliser HisA electrostatic stabiliser LysNone A electrostatic stabiliser LysA metal ligand AsnA metal ligand GluA metal ligand HisA activator, increase nucleophilicity, proton acceptor LysA proton acceptor Chemical Components ingold: bimolecular electrophilic addition , ingold: bimolecular nucleophilic addition , proton transfer.
Catalytic Residues Roles Residue Roles LysA electrostatic stabiliser AsnA electrostatic stabiliser HisA electrostatic stabiliser LysNone A electrostatic stabiliser LysA metal ligand AsnA metal ligand GluA metal ligand LysA proton donor Chemical Components native state of enzyme is not regenerated , overall product formed , assisted keto-enol tautomerisation , proton transfer.
Contributors Mei Leung, Gemma L. Holliday, James Willey. Its carbonyl oxygen accepts H-bond from carbamate NH, increasing the positive charge on this nitrogen. It reacts with CO2 to form carbamate which provides monodentate ligand to Mg II to complete the active site.
0コメント