Activity expressed from cloned Anacystis nidulans large and small subunit ribulose bisphosphate carboxylase genes

Summary The ribulose bisphosphate carboxylase/oxygenase (EC4.1.1.39) (RubisCO) large and small subunit genes from Anacystis nidulans have been cloned as a single fragment into M 13mp10 and pEMBL8 and expressed in Escherichia coli. From M 13mp10 a low yield of enzyme with high specific activity was obtained. The molecular weight of the active enzyme was 260 000 Da and of the inactive enzyme approximately 730 000 Da. The small and large subunits cloned separately did not express activity. The RubisCO gene cloned into pEMBL8 expressed activity up to 22 times that from the M 13 cloned RubisCO DNA. The RubisCO protein produced by the pEMBL cloned gene had a normal MW (550 000). Immunoprecipitation and polyacrylamide gel electrophoresis showed the presence of both large and small subunits.     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Protein degradation in lemna with particular reference to ribulose bisphosphate carboxylase: I. The effect of light and dark

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modification of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.     

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis

The functions of His²⁹¹, His²⁹⁵ and His³²⁴ at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His²⁹¹ by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His³²⁴ (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His²⁹⁵, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated K_m(RuBP) values and unchanged CO₂/O₂ specificity factors compared to recombinant wild-type.Abbreviations CABP D-2-carboxyarabinitol 1,5 bisphosphate - IPTG isopropyl-b-d-thiogalactopyranoside - L large subunit of rubisco - PAGE polyacrylamide gel electrophoresis - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-P₂, ribulose 1,5 bisphosphate - S small subunit of rubisco - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol     

Biochemical Specialization of Photosynthetic Cell Layers and Carbon Flow Paths in Suaeda monoica

Suaeda monoica Frossk. ex J. F. Gmel is a C₄ plant with three different photosynthesizing cell layers. The outer chlorenchymatous layer shows a high activity of phosphoenolpyruvate (PEP) carboxylase but none of ribulose bisphosphate (RuBP) carboxylase. The electrophoretic protein band of RuBP carboxylase was missing in this layer. The second chlorenchymatous cells layer shows a very high activity of RuBP carboxylase and NAD malic enzyme and only traces of activity of PEP carboxylase. The third photosynthesizing cell type is comprised of the water tissue. It has moderate activities of RuBP carboxylase and PEP carboxylase. A model for carbon flow in Suaeda monoica leaves is proposed.     

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.     

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O(2)-Low, CO(2)-Free N(2)

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO₂-free air for 3 hours with either 1% O₂ in N₂ or N₂-only and then returned to normal air of 340 microliters per liter CO₂, 21% O₂ in N₂. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO₂-free treatments as was photosynthetic CO₂ fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N₂ only treatment. During the CO₂-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO₂-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO₂-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO₂ fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO₂ fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO₂ and Mg²⁺. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand.     

Mutations in a sequence near the N-terminus of the small subunit alter the CO2/O2specificity factor for ribulose bisphosphate carboxylase/oxygenase

Seven unique substitutions have been introduced by site-directed mutagenesis into the first conserved region of the small subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans 6301. After expression of each large, altered-small subunit gene tandem in Escherichia coli, two substitutions in the small subunit tyr17asp17 (Y17D) and arg10gly10 (R10G) result in little or no carboxylase activity. For the latter substitution, no L₈S₈enzyme complex could be detected suggesting that this mutation prevents assembly. Mutant enzymes containing the following substitutions R11G, T14A, S16A, Y17D and P19A have CO₂/O₂specificity factors ( values) of 40, 35, 18, 39 and 44, respectively, compared with that of 44 for wild-type recombinant enzyme whereas P20A has full carboxylase activity and a value of 55.     

Cloning,expression and directed mutagenesis of the genes for ribulose bisphosphate carboxylase/oxygenase

The dominant natural form of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of large (L) 55-kDa and small (S) 15-kDa subunits. This enzyme (as the L₈S₈ form) is widely distributed among oxygenic photosynthetic species and among chemosynthetic bacteria. Another form lacking small subunits is found as an L₂ dimer in Rhodospirillum rubrum or an L oligomer of uncertain aggregation state from Rhodopseudomonas spharoides. The present article reviews two basically different approaches in cloning the R. rubrum gene for RuBisCO. One results in high level expression of this gene product fused with a limited aminoterminal stretch of -galactosidase and the other results in expression of wild-type enzyme in Escherichia coli. Also reviewed are a number of reports of cloning and assembly of the L₈S₈ enzyme in using E. coli L and S subunit genes from Anacystis nidulans, Anabaena 7120, Chromatium vinosum and Rps. sphaeroides.In vitro oligonucleotide-directed mutagenesis has been applied to the gene for RuBisCO from R. rubrum. In terms of contributing new information to our understanding of the catalytic mechanism for RuBisCO, the most significant replacement has been of lys 166 by a number of neutral amino acids or by arg or his. Results establish that lys 166 is a catalytically essential residue and illustrate the power of directed mutagenesis in understanding structure-function correlates for RuBisCO.Oligonucleotide-directed mutagenesis has also been applied to the first and second conserved regions of the S subunit gene for RuBisCO from A. nidulans. In the latter region, corresponding amino acid changes of trp 55 and trp 58 to phe, singly or together, had little or no effect upon enzyme activity. In contrast, mutagenesis in the first conserved region leading to the following pairs of substitutions: arg10 arg 11 to gly 10 gly11; thr14 phe 15 ser 16 to ala 14 phe 15 ala 16; ser 16 tyr 17 to ala 16 asp 17; or pro 19 pro 20 to ala 19 ala 20, are all deleterious.Advances are anticpated in the introduction and expression of interesting modifications of S (and L) subunit genes in plants. A new method of introducing and expressing foreign genes in isolated etiochloroplasts is identified.Abbreviations RuBisCO ribulose bisphosphate carboxylase/oxygenase - 2-CABP 2-carboxyarabinitol-1,5-bisphosphate - 4-CABP 4-carboxyarabinitol-1,5-bisphosphate     

Characteristics and Activity Changes of Proteolytic Enzymes in Apple Leaves during Autumnal Senescence

At least four different proteinases are present in senescing apple leaves (Malus domestica Borkh. cv. Golden Delicious) as determined by their pH optima, substrate specificity, and their reactivity to proteinase inhibitors. An enzyme active at pH 4.5 to 5.0 appears to be a sulfhydryl-dependent (iodoacetamide and phenylmercuric acetate-sensitive) endoproteinase, and degradation of the large subunit of ribulose bisphosphate carboxylase was observed only with this enzyme. It is tentatively concluded that this endoproteinase is responsible for the breakdown of ribulose bisphosphate carboxylase in vivo. However, the presence of more than one endoproteinase in apple leaves is suggested by the broad range of pH optima of the SH-dependent enzyme. Another enzyme active at pH 6.0 appears to be a carboxypeptidase, and was sensitive to phenylmethylsulfonylfluoride. This enzyme showed a strong hydrolytic activity against carbobenzoxyphenylalanylalanine. A sulfhydryl-dependent aminopeptidase and a second hydroxyl-dependent carboxypeptidase were active at pH 7.5

Total autolytic activity (the sulfhydryl-dependent endoproteinase) as measured by the disappearance of proteins decreased during the period of protein decline. Evidence is presented that the measured proteinase activity can be dependent on assay methods and substrates. While the disappearance of protein measures most of endo-type activity, the ninhydrin assay appears to measure exo-type activity preferentially.

     

Synthesis and assembly of bacterial and higher plant Rubisco subunits in Escherichia coli

The synthesis in Escherichia coli of both the large and small subunits of cereal ribulose bisphosphate carboxylase/oxygenase has been obtained using expression plasmids and bacteriophages. The level and order of synthesis of the large and small subunits were regulated using different promoters, resulting in different subunit pool sizes and ratios that could be controlled in attempts to optimize the conditions for assembly. Neither assembly nor enzyme activity were observed for the higher plant enzyme. In contrast, cyanobacterial large and small subunits can assemble to give an active holoenzyme in Escherichia coli. By the use of deletion plasmids, followed by infection with appropriate phages, it can be demonstrated that the small subunit is essential for catalysis. However, the small subunit is not required for the assembly of a large subunit octomer core in the case of the Synechococcus enzyme; self-assembly of the octomer will occur in an rbcS deletion strain. The cyanobacterial small subunits can be replaced by wheat small subunits to give an active enzyme in Escherichia coli. The hybrid cyanobacterial large/wheat small subunit enzyme has only about 10% of the level of activity of the wild-type enzyme, reflecting the incomplete saturation of the small subunit binding sites on the large subunit octomer, and possibly a mismatch in the subunit interactions of those small subunits that do bind, giving rise to a lower rate of turnover at the active sites.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - L large subunit - Rubisco ribulose bisphosphate carboxylase/oxygenase - S small subunit     

Photorespiration-induced reduction of ribulose bisphosphate carboxylase activation level

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C₃ crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O₂, 350 microliters per liter CO₂, balance N₂), but not in nonphotorespiratory conditions (2% O₂, 350 microliters per liter CO₂, balance N₂). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg²⁺ for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg²⁺ and pH but rather is associated with metabolite flux through the photorespiratory pathway.     

Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme.

Oligonucleotide-directed mutagenesis of cloned Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase with a synthetic 13mer oligonucleotide primer was used to effect a change at Met-330 to Leu-330. The resultant enzyme was kinetically examined in some detail and the following changes were found. The Km(CO2) increased from 0.16 to 2.35 mM, the Km(ribulose bisphosphate) increased from 0.05 to 1.40 mM for the carboxylase reaction and by a similar amount for the oxygenase reaction. The Ki(O2) increased from 0.17 to 6.00 mM, but the ratio of carboxylase activity to oxygenase activity was scarcely affected by the change in amino acid. The binding of the transition state analogue 2-carboxyribitol 1,5-bisphosphate was reversible in the mutant and essentially irreversible in the wild type enzyme. Inhibition by fructose bisphosphate, competitive with ribulose bisphosphate, was slightly increased in the mutant enzyme. These data suggest that the change of the residue from methionine to leucine decreases the stability of the enediol reaction intermediate.     

Purification of RuBisCO from the thermophilic cyanobacterium Synechococcus sp. strain a-1

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the key enzyme of the Calvin Benson cycle, has been purified from a thermophilic cyanobacterium, Synechococcus sp. strain a-1 and characterized. The enzyme is an L₈S₈-type hexadecamer with a molecular mass of 530 kDa. The enzyme was stable against heat treatment up to 70°C, which is the highest value among the RuBisCOs so far purified. The K_m value for ribulose bisphosphate on the carboxylase activity was substantially higher than those observed for RuBisCOs obtained from mesophilic autotrophs. The N-terminal amino acid sequence for the large subunit of the enzyme was highly similar to those of the other cyanobacteria despite the significant differences in heat stability.