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.     

The effects of light quality and intensity on the synthesis of ribulose-1,5-bisphosphate carboxylase and its mRNAs in the green alga Chlorogonium elongatum

In the green alga Chlorogonium elongatum the promoting effect of light on the synthesis of ribulose bisphosphate carboxylase/oxygenase (RuBPCase) is mainly caused by blue light of wavelengths between 430 nm and 510 nm, with a maximum effect at about 460 nm. Blue light also causes an increase in the amounts of the mRNAs for the large and the small subunits of the enzyme. Furthermore, the concentration of RuBPCase is affected by the light energy fluence rate. The rate of synthesis as well as the maximal obtainable concentration of the enzyme are functions of the light energy fluence rate up to 26 W·m^-2. No further increase occurs beyond that intensity. The quantity of irradiation also alters the concentrations of the subunit mRNAs. The results indicate that the changes in the mRNA levels are the major regulatory steps in the light-dependent synthesis of the RuBPCase enzyme.Abbreviations LSU large subunit - pSSU precursor of the small subunit - RuBPCase ribulose bisphosphate carboxylase/oxygenase EC 4.1.1.39 Dedicated to Prof. Dr. E. Bünning on the occasion of his 80 th birthday     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Ribulose bisphosphate carboxylase from a mutant strain of Chlamydomonas reinhardii deficient in chloroplast ribosomes

The ac-20 mutant strain of the unicellular green alga, Chlamydomonas reinhardii, lacks both chloroplast ribosomes and ribulose bisphosphate carboxylase activity when grown on organic medium. Under these conditions, the cells do not posses pools of either the large or small subunit of this enzyme. When transferred to inorganic medium, the carboxylase activity recovers. During this recovery, de novo synthesis of both subunits occurs. Synthesis of both subunits is inhibited by chloramphenicol even when possible free subunit pools rather than just the subunits incorporated into whole enzyme are examined.Abbreviations RubP ribulose bisphosphate - CAP D-threochloramphenicol - CHI cycloheximide - PPO 2,5-diphenyloxazole - POPOP 1,4-bis[2(5-phenyloxazolyl)]-benzene - SDS sodium dodecyl sulfate     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

A critical arginine in the large subunit of ribulose bisphosphate carboxylase/oxygenase identified by site-directed mutagenesis

Rapid inactivation by phenylglyoxal of ribulose bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) from the cyanobacterium Anacystis nidulans suggests the presence of an essential arginine, the modification of which is reduced in the presence of the substrate ribulose bisphosphate. Arginine 292 in the large subunit of ribulose-P2 carboxylase from A. nidulans was chosen for site-directed mutagenesis studies on the basis of the complete conservation of this residue in corresponding sequences of ribulose-P2 carboxylase from divergent organisms. Arginine 292 was changed to leucine and to lysine by directed mutagenesis using suitable plasmids and the bacteriophage M13. Both substitutions resulted in the production of purifiable holoenzyme with no activity after expression in Escherichia coli.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

The Rubisco subunit binding protein

Chloroplasts contain an abundant soluble protein that binds non-covalently newly synthesized large and small subunits of the enzyme ribulose bisphosphate carboxylase-oxygenase. This binding protein has been purified from Pisum sativum and Hordeum vulgare in the form of a dodecamer consisting of equal amounts of two types of subunit. These subunits are synthesized as higher molecular mass precursors by cytoplasmic ribosomes before import into the chloroplast. Antibodies raised against the purified binding protein from Pisum sativum detect polypeptides not only in extracts of plastids from several plant species but also in cell extracts of several bacterial species. The oligomeric binding protein dissociates reversibly into monomeric subunits in the presence of 1–5 mmol/liter MgATP. For one type of subunit the cDNA sequence has been isolated and determined and reveals homology with certain bacterial proteins.These observations are discussed in relation to the idea that the binding protein is an example of a general class of proteins termed "molecular chaperones" which are required for the correct assembly of certain oligomeric proteins such as the carboxylase from their subunits.Abbreviations BP Binding protein - Rubisco Ribulose bisphosphate carboxylase-oxygenase     

Subunit dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase from Chromatium vinosum

The large and small subunits of ribulose bisphosphate carboxylase from Chromatium vinosum were dissociated and separated at pH 9.6 by sucrose density gradient centrifugation. After further purification by gel filtration, the small subunit fraction contained no carboxylase activity. The large subunit fraction was highly depleted of small subunit based on analysis by denaturing polyacrylamide gel electrophoresis. Carboxylase activity of the large subunit fraction was approximately 1% of the untreated native enzyme. Addition of purified small subunit to the large subunit fraction yielded increases of up to 67-fold in carboxylase activity, further indicating that both subunit types are required for catalysis by this enzyme. The isolated large subunit was fully capable of high-affinity activator 14CO2 binding in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, indicating that the activator and catalytic sites were not grossly denatured by the depletion of small subunit. Kinetic constants of the native C. vinosum enzyme defined a new class of ribulose bisphosphate carboxylase, which permits the detection of possible kinetic differences if the large and small subunits can be favorably reassembled with those of another kinetic class. From experiments with the enzymes from tobacco and spinach leaves it is concluded that the enzyme from higher plant sources is not suitable for such dissociation/reconstitution-type experiments.     

Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported beta-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts

The large subunit of ribulose bisphosphate carboxylase from Anacystis nidulans 6301, and the β subunit of chloroplast ATP synthase from maize, were fused to the transit peptide of the small subunit of ribulose bisphosphate carboxylase from soybean. These proteins were assayed for post-translational import into isolated pea chloroplasts. Both proteins were imported into chloroplasts. Imported large subunits were associated with two distinct macromolecular structures. The smaller of these structures was a hybrid ribulose bisphosphate carboxylase holoenzyme, and the larger was the binding protein oligomer. Time-course experiments following import of the large subunit revealed that the amount of large subunit associated with the binding protein oligomer decreased over time, and that the amount of large subunit present in the assembled holoenzyme increased. We also observed that imported small subunits of ribulose bisphosphate carboxylase, although predominantly present in the holoenzyme, were also found associated with the binding protein oligomer. In contrast, the imported β subunit of chloroplast ATP synthase did not assemble into a thylakoid-bound coupling factor complex.     

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.     

Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270

Acidithiobacillus ferrooxidans ATCC 23270 possesses two copies of form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The nucleotide sequence identity between the two large and two small subunit peptides was 75% and 58%, respectively. It is proposed that the two copies resulted from lateral gene transfer. Received: 27 October 2000 / Accepted 7 December 2001     

A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli

Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220–224], was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme. This construction entailed removing all lacZ-coding sequences and a portion of the 5'-noncoding leader of the R. rubrum rbc gene. The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R. rubrum ribosome binding site and the rbc structural gene. The carboxylase expressed in E. coli JM107 was purified to near homogeneity and, based on subunit M_r and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R. rubrum. N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same.     

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.     

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.     

Plastid targeting of E. coli β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells

Summary Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target -glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.Abbreviations BMS maize Black Mexican Sweet suspension culture cells - CTP chloroplast targeting peptide - glgC16 an allosteric variant of E. coli ADP-glucose pyrophosphorylase - GUS -glucuronidase - LUX luciferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSU small subunit of ribulose bisphosphate carboxylase     

Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [¹⁴C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [¹⁴C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.     

Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.     

The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.