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.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: I. Inducible formation

The nature of the inducible formation of enzymes engaged in the photosynthetic CO₂ fixation was examined in Chromatium vinosum during its autotropic development. Although the activity of RuBP carboxylase was the lowest among several enzyme activities examined, it was enhanced 2.5 times during a 5-hr incubation, while other enzyme activities were little altered. The enhancement of the RuBP carboxylase activity was dependent on the presence of reduced sulfur compounds in the incubation medium and illumination (>100 lx). The increase in enzyme activity, however, was repressed by CO₂ or pyruvate. Furthermore, O₂ markedly reduced the enzyme activity. In order to prove whether or not the enhancement of RuBP carboxylase activity was attributable to the biosynthesis of the enzyme, the incorporation of [³⁵S]methionine into RuBP carboxylase was followed by immunoprecipitation analysis. The incorporation was dependent on the reduced sulfur compounds, and was repressed by elevating the CO₂ level.     

Distinct properties of Escherichia coli products of plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase directed by two sets of genes from the photosynthetic bacterium Chromatium vinosum

We have recently described the existence of two sets of genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase), rbcA-rbcB and rbcL-rbcS, in the photosynthetic purple sulfur bacterium Chromatium vinosum (Viale, A.M., Kobayashi, H., and Akazawa, T. (1989) J. Bacteriol. 171, 2391-2400). These genes were cloned in plasmid vectors, and their expression was studied in Escherichia coli. Expression of rbcA-rbcB in E. coli was obtained under the control of its own promoter. On the other hand, expression of rbcL-rbcS in this host was not observed unless these genes were cloned under the control of the tac promoter. Purified rbcA-rbcB and rbcL-rbcS products from E. coli consisted of large and small subunits in equimolar ratios. They also showed very close elution profiles to Rbu-P2 carboxylase isolated from C. vinosum in size-exclusion chromatography columns, thus suggesting hexadecameric (L8S8) structures. Vmax of Rbu-P2 carboxylase were very similar for both enzymes, but the Km values for CO2 and ribulose 1,5-bisphosphate showed some differences. Immunochemical and N-terminal amino acid sequence analyses of the large and small subunits encoded by rbcA-rbcB and rbcL-rbcS also differed, especially at the level of the small subunits. The comparisons described above as well as the analysis of C. vinosum crude extracts by anion-exchange chromatography indicated that Rbu-P2 carboxylase encoded by rbcA-rbcB was the only species detected in the photosynthetic bacterium.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: II. Biosynthesis of constituent subunits

[³⁵S]Methionine-labeled free subunits A and B of RuBP carboxylase were present in barely detectable amounts; the radioactivity in the free subunit B was approximately 1/150th of that in the subunit B contained in the holoenzyme of RuBP carboxylase. The turnover rates of subunits A and B in the holoenzyme were equal at each time during the incubation period. The ratio of subunit A to subunit B was constant throughout the incubation time both in quantity and in the level of [³H]leucine and [³⁵S]methionine incorporated. CO₂ contained in the incubation medium suppressed [³⁵S]methionine incorporation into both subunits A and B equally. These results suggest that the biosynthesis of subunits A and B is completely synchronized and may be regulated by identical mechanisms.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium, Chromatium vinosum. III. Absence of extrachromosomal DNA

Inducible formation of ribulose-1,5-bisphosphate (RuBP) carboxylase in the cells of Chromatium vinosum under autotrophic conditions was not affected by six different inhibitors of DNA synthesis. Photosynthetic CO2 fixation and RuBP carboxylase activities were not influenced by seven reagents known to eliminate plasmids. Plasmids were not detectable by agarose gel electrophoresis employing either the cleared lysate or alkaline sodium dodecyl sulfate method, nor were they detected by ethidium bromide-CsCl density gradient centrifugation. Overall experimental results tend to indicate that plasmids are absent in the Chromatium cells and that the induction of RuBP carboxylase is presumably not regulated in the DNA replication process.     

Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions

Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg²⁺ and HCO₃⁻, and then stored for up to 30 days at 4 or −18°C or in liquid N₂. Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg²⁺ and CO₂ and subsequent storage at either 4°C or in liquid N₂ or flushing the samples with N₂ and rapid freezing and storage in liquid N₂ are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved.     

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.     

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.     

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

Phylogeny and evolution of the ribulose 1,5-bisphosphate carboxylase/oxygenase genes in prokaryotes

The review considers the phylogeny and evolution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is the key enzyme of the autotrophic Calvin-Benson cycle and the most abundant protein on Earth. RuBisCO occurs in several structural and functional forms, including fully functional forms I, II, and III, which catalyze carboxylation/oxygenation of ribulose 1,5-bisphosphate, and RuBisCO-like form IV, which lacks carboxylating activity. The genomic localization, operon structure, and copy number of the RuBisCO genes vary among different autotrophic organisms. The RuBisCO gene phylogeny substantially differs from the phylogeny of other conserved genes, including the 16S rRNA gene. The difference is due to duplication/deletion and horizontal gene transfer events that were common in the evolution of autotrophic organisms.     

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.     

Nuclear DNA regulates the level of ribulose 1,5-bisphosphate carboxylase oxygenase in Medicago sativa L.

Summary The response to selection for leaf proteins was studied during three selection cycles. Selection for high total nitrogen content showed 75% heritability, and the levels of both ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) and cytoplasmic protein were strongly under nuclear DNA control. High and low protein content were correlated with chloroplast area. Although the amounts of nuclear DNA were similar, the ratio of Rubisco/DNA and chlorophyll/DNA changed during the selection process. It can be concluded that the levels of Rubisco achieved in mature plants of M. sativa are under nuclear DNA control. The possible involvement of small subunit (SSU) genes in controlling these levels is discussed.     

Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.     

Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteolytic susceptibility of the native CO(2)-fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was reproduced with Rubiscos from other algae and higher plants, as well as with other chemical modifications of protein cysteines. N-terminal sequencing of the fragments showed that band I arised from clipping the unstructured N-terminal stretch of the large subunit up to Lys18. Band II was generated by a cleavage close to Val69. The increased susceptibility of the oxidized form resulted from proteases gaining access to a loop (from Ser61 to Thr68) located between stretches of secondary structure that form the N-terminal domain. Native electrophoresis and kinetic analysis of fragment accumulation during subtilisin digestion demonstrated that subunit dissociation was induced by the proteolytic processing at the Ser61-Thr68 loop, which is characteristic of the oxidized Rubisco. Holoenzyme dissasembly was readily followed by the full degradation of the released subunits. In contrast, the limited processing to band I observed with the reduced enzyme did not compromise the quaternary structure of the Rubisco hexadecamer, thus preventing further proteolysis.     

Phylogeny and functional expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI.

The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO(2) by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the beta and gamma subgroups of the class Proteobacteria are also presented. All except one of the beta-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other beta-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.