D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO2/Mg2+-activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 mM and a maximum pseudo-first-order rate constant of 7 X 10(-3) s-1. The rate constant for reactivation was 1.2 X 10(-2) s-1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme X CO2 X Mg2+ X CABP.     

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.     

Differential effects of metal ions on Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase and stoichiometric incorporation of HCO3- into a cobalt(III)--enzyme complex.

Mg2+ or Mn2+ ions supported both the carboxylase and oxygenase activities of the Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase. For the carboxylase reaction, Mn2+ supported 25% of the maximum activity obtained with Mg2+; oxygenase activity, however, was twice as great with Mn2+ as compared to that with Mg2+. A further differential effect was obtained with Co2+. Co2+ did not support carboxylase activity and, in fact, was a strong inhibitor of Mg2+-dependent carboxylase activity, with a Ki of 10 microM. Co2+ did, however, support oxygenase activity, eliciting about 40% of the Mg2+-dependent oxygenase activity. No other divalent cations supported either activity. With high concentrations of Mg2+ or Mn2+, maximum carboxylase activity was seen after a 5-min activation period; activity decreased to about half of maximum after 30-min activation. A similar time dependence of activation was observed with Mn2+-dependent oxygenase activity but was not seen for Mg2+- or Co2+-dependent activity. Both carboxylase and oxygenase activities were inactivated by the oxidation of Co2+ to Co(III) with the resultant formation of a stable Co(III)--enzyme complex. In the presence of HCO3- (CO2), Co(III) modification was stoichiometric, with two cobalt atoms bound per enzyme dimer. Carbon dioxide was also incorporated into this Co(III)--enzyme complex, but only one molecule per enzyme dimer was bound, indicative of half-the-sites activity. These results thus indicate that there are substantial differences in the metal ion sites of the carboxylase and oxygenase activities of R, rubrum ribulosebisphosphate carboxylase/oxygenase.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase

Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme.     

Characterization of the oxygenase activity in a mutant of Chlamydomonas reinhardi exhibiting altered ribulosebisphosphate carboxylase.

A previously described Mendelian mutant of Chlamydomonas reinhardi, ac i72, exhibiting altered ribulosebisphosphate carboxylase activity and unable to grow on minimal medium is examined for changes in ribulosebisphosphate oxygenase activity. The ribulosebisphosphate oxygenase activity of the enzyme purified from both wild type and ac i72 is compared over a pH range from 7.0 to 9.5. Both enzymes exhibit maximum activity at pH 9.0. However, the ac i72 enzyme is twice as active as the wild type enzyme at a physiological pH of 7.0. The studies in vivo of the products of CO2 fixation of ac i72 and wild type cells in the presence of high and low O2 concentration shows that due to a lower level of carboxylation, the ac i72 cells fix CO2 at half the rate of wild type cells. In ac i72, 24% of the photosynthetically fixed 14C is channelled into the water-soluble fraction as opposed to 6% in wild type. Thin-layer chromatography of the water-soluble fraction showed extensive accumulation of components of the glycolate pathway in ac i72 as compared to wild type. This indicates that the oxygenase activity of the enzyme prevails in ac i72 in vivo. Since a high concentration of glycolate is toxic to cells of C. reinhardi, the high oxygenase activity of ac i72 provides an explanation for the inability of ac i72 to grow phototrophically even though its rate of CO2 fixation is half that of wild type. This toxicity to glycolate is overcome by growth under amber illumination or low O2 concentration.     

Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum

The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.     

Ribulose 1, 5-Bisphosphate Carboxylase/Oxygenase Activity and Photorespiration during the Ageing of Flag Leaves of Wheat

Photosynthesis, photorespiration, and ribulose bisphosphatecarboxylase/oxygenase activities were measured in intact flagleaves of wheat during ageing. Photorespiration declined verylittle as the leaves aged, and much less than photosynthesis.These changes could not be explained by changes in the ribulosebisphosphate carboxylase to oxygenase ratio of fraction 1 protein.As the leaves grew older the enzyme activities in extracts ofleaves declined in parallel so the ratio remained constant.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum

The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits. Truncations of the R. rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme. A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate-saturated catalytic rate resembled that of the wild-type enzyme but which had weaker affinities for ribulose-P2 and CO2.     

Carbon isotope (13C/12C) effect of photorespiration in photosynthetic organisms. Evidence for existence, probable mechanism

Experimental evidence in favor of the new phenomenon predicted for photosynthesizing organisms, the fractionation of carbon isotopes in photorespiration is presented. A possible mechanism of this process is discussed. The fractionation of carbon in isotopes photorespiration occurs in the oxygenase phase of the functioning of ribulosebisphosphate carboxylase/oxygenase (rubisco), the key enzyme of photosynthesis, which is capable to act as carboxylase and oxygenase. Which function of the enzyme is active depends on CO2/O2 concentration ratio, which periodically changes in a cell. The key reaction in the mechanism of carbon isotope fractionation in photorespiration is glycine decarboxylation, which results in the splitting and removal from the cell of CO2 enriched with 12C and the accumulation of 13C photorespiratory carbon flow. The coupling of photorespiration and CO2 photoassimilation gives rise to two isotopically different carbon flows, which fill up separate carbohydrate pools, which are the sources of carbon in the following syntheses in the dark phase of photosynthesis. This enables one to identify, from the carbon isotope ratio of metabolites, their involvement in the photorespiratory and assimilatory carbon flows, to investigate the pathways of carbon metabolism, and to estimate more thoroughly the biosynthetic role of photorespiration.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Thermal properties and oxygenase activity of ribulose-1,5-bisphosphate carboxylase from the thermophilic purple bacterium, Chromatium tepidum

Abstract Ribulose-1,5-biphosphate carboxylase (RuBPCase) partially purified from the thermophilic purple bacterium Chromatium tepidum displayed maximum carboxylase activity at 50°C, while enzyme from a related mesophilic species, Chromatium vinosum , was completely inactive at 50°C. RuBPCase from C. tepidum showed ribulose-1,5- bisphosphate-dependent oxygenase activity, and, in addition, O₂ was found to partially destroy carboxylase activity. It is concluded that thermophilic purple bacteria produce heat-stable RuBPCase and that all RuBPCases, even those from an obligate anaerobe such as C. tepidum , have associated oxygenase activity.     

Formation of the small subunit in the absence of the large subunit of ribulose 1,5-bisphosphate carboxylase in 70 S ribosome-deficient rye leaves.

Immunological tests with monospecific antisera to ribulosebisphosphate carboxylase (EC 4.1.1.39) and to its large and small subunits indicated the presence of a protein with antigenic properties of the small subunit in the absence of the large subunit in the leaves of young rye plants (Secale cereale L.) with a high-temperature-induced (32 °C) deficiency of 70 S plastid ribosomes. The small subunit-like protein was isolated from crude extracts of plastid ribosome-deficient 32 °C-grown leaf tissue by the use of columns with immobilized antibody. The main polypeptide retained by the immobilized antibodies had the same mobility after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as the small subunit of ribulosebisphosphate carboxylase and was also immunologically identical to the small subunit. The small subunit-like protein was present in the supernatant as well as in the membrane fraction of isolated 70 S ribosome-deficient plastids. At very young stages of normal leaves grown at a permissive temperature (22 °C) an excess of small subunit was observed that was also not integrated into the complete ribulosebisphosphate carboxylase molecule. From the results, we conclude that the synthesis of the small subunit occurs on cytoplasmic ribosomes and is not strictly coordinated with the translation of the large subunit in the chloroplast. During early leaf development, the formation of the large subunit seems to be the ratelimiting step in the synthesis of ribulosebisphosphate carboxylase.     

Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking in digests of the substrate-protected enzyme. Peptides representing two sites of modification have been obtained from the inactivated carboxylase. Both sites of reaction have been identified as lysyl residues based on the conversion of the derivatives to free lysine by oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel electrophoretic experiments show that both essential lysyl residues are contained within the large subunit of ribulosebisphosphate carboxylase. In addition to lysyl residues, sulfhydryl groups of the carboxylase are also modified, but their modification seems to play little role in the inactivation process. The carboxylase modified in the presence of substrate contains sulfhydryl derivatives but is essentially lacking in lysyl derivatives. By comparing the profiles from ion exchange chromatography of labeled peptides in digests of inactivated and substrate-protected enzyme, we conclude that the same sulfhydryl groups are modified in the absence and presence of substrate.     

Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis

Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.