Role of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in the activation process

The large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from the cyanobacterium Aphanothece halophytica and from the purple sulfur photosynthetic bacterium Chromatium vinosum (strain D) were separated by sucrose density gradient centrifugation at low ionic strength and alkaline pH (9.3), respectively. It was found that subunit B enhances the extent of activation by CO2 and Mg2+ at equilibrium of the two homologous enzymes consisting of Aphanothece large subunit and its own small subunit (AaBa) and the Chromatium large subunit and its own small subunit (AcBc). The extent of activation induced by saturating amounts of subunit B was larger with AcBc than AaBa, amounting to 3.7- and 1.8-fold of that by each catalytic core alone, respectively. Subunit B stimulated both the extent of activation at equilibrium and catalysis in a parallel and simultaneous manner with respect to the concentration of B in both homologous enzymes. These results suggest that subunit B interacts with both activation and catalytic sites simultaneously. On the other hand, Chromatium subunit B only slightly stimulated the extent of activation in the hybrid enzyme AaBc. The role of subunit B in enhancing the extent of activation at equilibrium can be substituted by the effect exerted by 6-phosphogluconate. Both homologous enzymes AaBa and AcBc showed a faster deactivation rate when the enzyme was activated in the absence of subunit B. The mechanism by which subunit B promotes activation seems to involve its effect on stabilizing the activated enzyme molecule. From studies on the Km for substrate CO2 in the hybrid enzyme AaBc a major involvement of subunit B in influencing Km (CO2) seems unlikely.     

Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition

A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.     

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.     

A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor.

Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301. The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E. coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes. Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively. Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP. The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate. Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO. Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins. These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.     

Interaction of constituent subunits in ribulose 1,5-bisphosphate carboxylase from Aphanothece halophytica

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) from the halophilic cyanobacterium, Aphanothece halophytica, dissociates into catalytic core (large subunit A oligomer) and small subunit B under low ionic strength during sucrose density gradient centrifugation. Supplementation of KCl, NaCl, or K2SO4 ( [I] = 0.3 M) partly prevents the dissociation, the preventive effect of divalent cation salts such as MgCl2 and CaCl2 being more effective than monovalent cation salts. RuBisCO with its higher-plant-type molecular form can be isolated from the cyanobacterial extracts using gradient medium containing 0.3 M KCl, 20 mM MgCl2, and 10 mM CaCl2. The isolated enzyme contains large subunit A and small subunit B in a molar ratio of approximately 1:1, estimated from the densitometric scanning of Coomassie blue-stained gels. During the second sucrose density gradient centrifugation to remove minor contaminants, a small amount of subunit B is depleted from the holoenzyme. Determination of the molecular weight by equilibrium centrifugation and electron microscopic observation have confirmed that the cyanobacterial RuBisCO has an A8B8-type structure. The enzyme activity per se is found to be sensitive to concentrations of salts, and small subunit B is obligatory for the enzyme catalysis. It has been shown that the more the enzyme activity is inhibited by salts, the tighter the association of subunit B becomes. It is likely that the active enzyme retains the loose conformational structure to such an extent that the dissociable release of subunit B from the holoenzyme in vivo is not allowed.     

Factors affecting the dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase/oxygenase from Aphanothece halophytica

Factors affecting the mutual interaction between the catalytic core [octamer of large subunit (A)] and the small subunit (B) comprising ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from the superhalophilic cyanobacterium, Aphanothece halophytica, were investigated. The enzyme molecule dissociated into the catalytic core highly depleted of subunit B and the monomeric form of subunit B during density gradient centrifugation (15 h, 4 degrees C) in a sucrose solution of low ionic strength ([I] less than or equal to 50 mM), whereas dissociation was effectively prevented in the presence of 0.3 M KCl. Under the latter condition, dissociation of the enzyme molecule was almost completely prevented by raising the temperature to 20 degrees C, suggesting hydrophobic interaction between catalytic core and subunit B. The addition of RuBP to the sucrose gradient was shown to effectively reduce the molecular dissociation, suggesting a close interaction between the catalytic site and the binding site of subunit B with the catalytic core directly or indirectly. The dissociation was accelerated at alkaline pH higher than 8.5. Reconstitution of the enzymatically active molecular form from the separated components, catalytic core highly depleted of subunit B and B1, was done under various conditions. Both carboxylase and oxygenase activities increased proportionately with the amount of subunit B and then became saturated. From the reconstitution kinetics of RuBP carboxylase, the binding constant of subunit B (KD) was estimated to be about 30 nM in the presence of bovine serum albumin under the usual assay conditions at pH 7.5 and 25 degrees C, but decreased to about 1 nM by the further addition of 0.3 M KCl. Alkaline pH (8.5 or 9) could increase KD by one order of magnitude. High KD was also observed as a result of lowering the temperature; however, the presence of 0.3 M KCl or 0.4 M sucrose or glycerol could effectively decrease the KD at low temperature from 900 nM to less than 50 nM. All these data indicate that the enzyme dissociation at low temperature can be prevented in vivo by cellular components such as salts, polyols, and substrate RuBP besides a factor of enzyme concentration.     

Ribulose 1,5-bisphosphate carboxylase from the halophilic cyanobacterium Aphanothece halophytica

Various structural and functional properties of ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) isolated from the halophilic cyanobacterium (blue-green alga) Aphanothece halophytica were reexamined. The ready dissociation of this algal RuBisCO during sedimentation in a linear sucrose density gradient was observed. Low NaCl concentrations promote the dissociation of small subunit (B) from the original native enzyme molecule as evidenced by the sucrose density gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is thus possible that the intracellular osmoticum of A. halophytica might influence the structural integrity and activity of RuBisCO. The low residual carboxylase activity ascribed to the catalytic core, an oligomer form of the large subunit (A) apparently deficient in small subunit (B), was found to be markedly stimulated by a protein component which appears identical to subunit B. The purification and structural characterization of the catalytic core and subunit B were attempted by step-wise column chromatography on DEAE-cellulose, Utrogel AcA 34, Sephadex G-75, and hydroxylapatite, and at the final stage each component was purified to near homogeneity, although the catalytic core is still associated with a small quantity of subunit B. The addition of subunit B to the catalytic core does not alter the K_m (HCO₃^?, RuBP) values, but V_max values are markedly enhanced. Sucrose density gradient centrifugation gave a value of 16 S for the catalytic core. The molecular weights of the monomeric forms of the catalytic core (subunit A) and subunit B were 5.0 × 10⁴ and 1.4 × 10⁴, respectively.     

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.     

Further studies on the subunit structure of Chromatium ribulose-1,5-phosphate carboxylase.

Upon alkali exposure Chromatium ribulose-1,5-bisphosphate carboxylase dissociates into constituent subunits, a catalytic oligomer of the larger subunit, A8, and monomeric form of the small subunit B. By sedimentation equilibrium molecular weights of the native enzyme and the catalytic oligomer produced by an alkali treatment were estimated to be 5.11 x 10 5 and 4.29 x 10 5, respectively. To provide information on reversibility of the dissociation by determining whether the enzymically inactive small subunit B of the whole enzyme molecule did indeed exchange with exogenously added subunit B a radioisotopic method was used. After initial alkaline dialysis at pH 9.2 of a mixture of a nonlabeled native enzyme preparation and 14C-labeled subunit B, and the subsequent dialysis at pH 7.0, incorporation of 14C into the recovered native enzyme was determined. Without the alkaline treatment there was no detectable exchange, while after alkaline dialysis for 5 and 10 hr the subunit B exchange was 89 and 82%, respectively. Rabbit antiserum prepared against the catalytic oligomer of the spinach ribulose-1,5-bisphosphate carboxylase, anti-(A) (spinach), inhibited the Chromatium carboxylase and oxygenase activities. This result together with the identical immunoprecipitation lines on an agar plate formed between the antiserum and the Chromatium carboxylase and between the antiserum and the catalytic subunit of the Chromatium enzyme strongly indicated structural near identity of the catalytic subunits of the spinach and Chromatium carboxylase molecules. Results also show that the catalytic site of the Chromatium ribulose-1,5-bisphosphate carboxylase and oxygenase exists in the large polypeptide chain.     

Biochemical characteristics of photosynthetic response to various external salanities in halotolerant and fresh water cyanobacteria

Abstract The effect of various salinities on photosynthetic carbon fixation, activities of carboxylating enzymes, and contents of chlorophyll and protein, was examined in a halotolerant ( Aphanothece halophytica ) and fresh water ( Aphanothece stagnina ) cyanobacterium. These parameters were quantified immediately and after 48 h incubation in altered salinities. Photosynthetic carbon fixation was lower at altered salinities than in controls in both species. A. halophytica with 48 h incubation showed increased carbon fixation with increasing NaCl concentration, whereas A. stagnina did not. Chlorophyll content was drastically reduced for all salinities in A. stagnina but was unchanged in A. halophytica . d -ribulose-1,5-bisphosphate carboxylase (RuBisCO) activity from A. halophytica increased at higher external salinities but decreased in cells of A. stagnina . In vitro, the sensitivity of RuBisCO to NaCl from both the species was similar. It was noted that in A. stagnina NaCl affected chlorophyll content, carbon fixation, and RuBisCO activity in decreasing order.     

A homolog of ribulose bisphosphate carboxylase/oxygenase-binding protein in Chromatium vinosum

A 700-kDa protein composed of 12 apparently identical 60-kDa subunits copurifies with the L8S8 form of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Chromatium vinosum. Chromatography on DEAE-Sephadex A-50 separates the two proteins in pure form. On the basis of the highly reproducible copurification and reaction of the 700-kDa protein with antibodies to pea RuBisCO large (L)-subunit-binding protein, the protein from C. vinosum is designated as a putative binding protein (PBP) for RuBisCO. Also the N-terminal sequence of PBP is quite similar to that of both alpha and beta subunits of the L-subunit-binding protein. Our present research suggests that PBP may be a RuBisCO small-subunit-binding protein in C. vinosum. Measurements of RuBisCO activity and of species that immunologically cross react with RuBisCO or PBP (by enzyme-linked immunosorbent assay) establish that levels of the two proteins vary together in C. vinosum grown on different carbon sources.     

Catalytically active hybrids formed in vitro between large and small subunits of different procaryotic ribulose bisphosphate carboxylases

Ribulose bisphosphate carboxylase from the procaryotic green alga, Prochloron (the symbiont of Lissoclinum patellum), has eight large and eight small subunits, and a low affinity for CO2, similar to that of cyanobacterial carboxylases. The small subunits were progressively removed from this carboxylase and from that from the cyanobacterium, Synechococcus ACMM 323, by twice-repeated, mild-acid precipitation. This procedure produced large-subunit octamers, greatly depleted in small subunits, as well as isolated small subunits. Catalytic activity of the large-subunit preparations reflected their residual small-subunit content. The two large-subunit preparations were reconstituted with both homologous and heterologous small subunits. The reassembled enzymes were catalytically competent in all cases. When fully saturated with small subunits, the hybrid enzymes were only about 20% less active than the homologously reconstituted enzymes. Heterologous reconstitution underscores the essential function of the small subunits in catalysis.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits.     

Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase

The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B. Lee and F. R. Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits. Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate. This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits. In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies. The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.     

Essentiality of the small subunit (B) in the catalysis of RuBP carboxylase/oxygenase is not related to substrate-binding in the large subunit (A)

The small subunit (B) of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase from Aphanothece halophytica is absolutely required for the catalysis, but depletion of subunit B does not significantly affect the formation of the quaternary complex-[enzyme.activator CO2.Mg.carboxyarabinitol bisphosphate] in the catalytic core. The inhibition of RuBP carboxylase activity by the reaction of the epsilon-amino group of a lysine in the RuBP-binding site with pyridoxal 5-P is the same whether subunit B is added to the catalytic core before or after the inactivating reaction. The function of subunit B is not related to the substrate binding.     

Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota

In early biological evolution anoxygenic photosynthetic bacteria may have been established through the acquisition of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The establishment of cyanobacteria may have followed and led to the production of atmospheric oxygen. It has been postulated that a unicellular cyanobacterium evolved to cyanelles which were evolutionary precursors of chloroplasts of both green and non-green algae. The latter probably diverged from ancestors of green algae as evidenced by the occurrence of large (L) and small (S) subunit genes for Rubisco in the chloroplast genome of the chromophytic algae Olisthodiscus luteus. In contrast, the gene for the S subunit was integrated into the nucleus in the evolution of green algae and higher plants. The evolutionary advantages of this integration are uncertain because the function of S subunits is unknown. Recently, two forms of Rubisco (L8 and L8S8) of almost equivalent carboxylase and oxygenase activity have been isolated from the photosynthetic bacterium Chromatium vinosum. This observation perpetuates the enigma of S subunit function. Current breakthroughs are imminent, however, in our understanding of the function of catalytic L subunits because of the application of deoxyoligonucleotide-directed mutagenesis. Especially interesting mutated Rubisco molecules may have either enhanced carboxylase activity or higher carboxylase:oxygenase ratios. Tests of expression, however, must await the insertion of modified genes into the nucleus and chloroplasts. Methodology to accomplish chloroplast transformation is as yet unavailable. Recently, we have obtained the first transformation of cyanobacteria by a colE1 plasmid. We regard this transformation as an appropriate model for chloroplast transformation.     

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.     

The structure of isolated Synechococcus strain WH8102 carboxysomes as revealed by electron cryotomography

Carboxysomes are organelle-like polyhedral bodies found in cyanobacteria and many chemoautotrophic bacteria that are thought to facilitate carbon fixation. Carboxysomes are bounded by a proteinaceous outer shell and filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the first enzyme in the CO(2) fixation pathway, but exactly how they enhance carbon fixation is unclear. Here we report the three-dimensional structure of purified carboxysomes from Synechococcus species strain WH8102 as revealed by electron cryotomography. We found that while the sizes of individual carboxysomes in this organism varied from 114 nm to 137 nm, surprisingly, all were approximately icosahedral. There were on average approximately 250 RuBisCOs per carboxysome, organized into three to four concentric layers. Some models of carboxysome function depend on specific contacts between individual RuBisCOs and the shell, but no evidence of such contacts was found: no systematic patterns of connecting densities or RuBisCO positions against the shell's presumed hexagonal lattice could be discerned, and simulations showed that packing forces alone could account for the layered organization of RuBisCOs.