Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

The oxygenase activity of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Catalysis by pure ribulose bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, which is a dimer (MW: 114,000) lacking small subunits, is inhibited by oxygen. Oxygen is a competitive inhibitor with respect to carbon dioxide. In the absence of carbon dioxide, the enzyme catalyzes the oxygenolytic cleavage of ribulose-1,5-bisphosphate with consumption of one mole of oxygen per mole of 3-phosphoglycerate produced.     

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.     

Preliminary structural studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Rhodospirillum rubrum has been crystallized in a form that is suitable for structural studies by x-ray diffraction. The asymmetric unit of the crystal contains one dimeric enzyme molecule of molecular mass 101,000 Da. The enzyme was activated prior to crystallization and is presumed to be in the CO2-activated state in the crystal. The method of hydrophobicity correlation has been used to compare the amino acid sequence of this molecule (466 residues) to that of the large subunit of a higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (477 residues in Nicotiana tabacum). The pattern of residue hydrophobicities is similar along the two polypeptides. This suggests that the three-dimensional folding of the large polypeptide chains may be similar in plant and bacterial enzymes. If this is so, knowing the structure of either the plant or bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase should aid in learning the structure of the other.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.     

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis

The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.     

Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate

The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.     

Truncation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum affects the holoenzyme assembly and activity.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site-directed mutagenesis to examine the role of the C-terminal tail section. Removal of the last and the penultimate alpha-helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild-type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end-to-end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2-carboxy arabinitol-1,5-bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C-terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.     

Bicarbonate inhibits ribulose-1,5-bisphosphate carboxylase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) rapidly extracted from leaves of wheat (Triticum aestivum) and purified activated RuBPCO were incubated in the presence and absence of 20 millimolar HCO₃⁻ and changes in activation state were followed. Rapid inactivation occurred in the presence, but not in the absence, of HCO₃⁻. Effects of CO₂ concentration and pH during preincubation before assay on activation state of RuBPCO were investigated in equilibrium studies. Twenty percent inactivation occurred at high CO₂ concentration if pH was high, but not if it was low, suggesting that RuBPCO was inactivated by HCO₃⁻. The inactivation by HCO₃⁻ was more rapid than the dissociation of activating CO₂ in CO₂-free buffer (both in the presence of 20 millimolar MgCl₂), suggesting that HCO₃⁻ was bound to the active enzyme complex. The dissociation of inactivating HCO₃⁻ from the enzyme was slow enough that inhibition could be demonstrated in experiments with HCO₃⁻ treatments during preincubation and constant conditions during assay. Inorganic phosphate did not seem to interfere with the binding of HCO₃⁻.     

Oxygen regulation of ribulose 1,5-bisphosphate carboxylase activity in Rhodospirillum rubrum.

The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of Rhodospirillum rubrum was exposed to atmospheric levels of oxygen. From 70 to 80% of the activity was lost within 12 to 24 h. Inactivation was apparent when the enzyme was assayed in situ (in whole cells) and when activity was measured in dialyzed crude extracts. The quantity of enzyme protein, as estimated from sodium dodecyl sulfate-polyacrylamide gels or as quantified immunologically, did not decrease within 24 h of exposure to air. Following extended exposure to aerobic conditions (48 to 72 h), degradation of enzyme occurred. These results indicate that the inactivation of RuBPC/O in R. rubrum may be due to an alteration or modification of the preformed enzyme, followed by eventual degradation of the inactive enzyme. When shifted back to anaerobic conditions (under an argon atmosphere), the RuBPC/O activity increased rapidly. This increase appeared to be due to de novo synthesis of enzyme. The increase in activity was not observed when the culture was maintained in the dark or in the absence of a suitable carbon source. Thus, the oxygen-mediated inactivation of RuBPC/O appeared to be due to some form of irreversible modification. The cloned R. rubrum RuBPC/O gene, expressed in Escherichia coli, yielded functional enzyme that was not affected by oxygen, indicating that inactivation in R. rubrum is mediated by a gene product(s) not found in E. coli.     

Role of asparagine-111 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as explored by site-directed mutagenesis.

Crystallographic studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum suggest that active-site Asn111 interacts with Mg2+ and/or substrate (Lundqvist, T., and Schneider, G. (1991) J. Biol. Chem. 266, 12604-12611). To examine possible catalytic roles of Asn111, we have used site-directed mutagenesis to replace it with a glutaminyl, aspartyl, seryl, or lysyl residue. Although the mutant proteins are devoid of detectable carboxylase activity, their ability to form a quaternary complex comprised of CO2, Mg2+, and a reaction-intermediate analogue is indicative of competence in activation chemistry and substrate binding. The mutant proteins retain enolization activity, as measured by exchange of the C3 proton of ribulose bisphosphate with solvent, thereby demonstrating a preferential role of Asn111 in some later step of overall catalysis. The active sites of this homodimeric enzyme are formed by interactive domains from adjacent subunits (Larimer, F. W., Lee, E. H., Mural, R. J., Soper, T. S., and Hartman, F. C. (1987) J. Biol. Chem. 262, 15327-15329). Crystallography assigns Asn111 to the amino-terminal domain of the active site (Knight, S., Anderson, I., and Br?ndén, C.-I. (1990) J. Mol. Biol. 215, 113-160). The observed formation of enzymatically active heterodimers by the in vivo hybridization of an inactive position-111 mutant with inactive carboxyl-terminal domain mutants is consistent with this assignment.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site

The X-ray structure of the quaternary complex of ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach with CO2, Mg2+ and a reaction-intermediate analogue (CABP) has been determined and refined at 2.4 A resolution. Cyclic non-crystallographic symmetry averaging around the molecular 4-fold axis and phase combination were used to improve the initial multiple isomorphous replacement phases. A model composed of one large subunit and one small subunit was built in the resulting electron density map, which was of excellent quality. Application of the local symmetry gave an initial model of the L8S8 molecule with a crystallographic R-value of 0.43. Refinement of this initial model was performed by a combination of conventional least-squares energy refinement and molecular dynamics simulation using the XPLOR program. Three rounds of refinement, interspersed with manual rebuilding at the graphics display, resulted in a model containing all of the 123 amino acid residues in the small subunit, and 467 of the 475 residues in the large subunit. The R-value for this model is 0.24, with relatively small deviations from ideal stereochemistry. Subunit interactions in the L8S8 molecule have been analysed and are described. The interface areas between the subunits are extensive, and bury almost half of the accessible surface areas of both the large and the small subunit. A number of conserved interaction areas that may be of functional significance have been identified and are described, and biochemical and mutagenesis data are discussed in the structural framework of the model.