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Query: UMLS:C1832526 (PCC)
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The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3'-truncated gene copy into the psb A-I locus. Both rbcL genes, in the psb A-I region and at the rbc locus, were non-functional; The former due to the 3' truncation, and the latter due to a deletion in the 5'-region (creating a 5' truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO2-requiring mutant ('cyanorubrum'). The 3' and the 5' truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L8S8) and R. rubrum (L2) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.
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PMID:Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination. 146 99

Pyruvate is a minor product of the reaction catalyzed by ribulosebisphosphate carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.
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PMID:Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase. 190 85

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.
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PMID:Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. 191 Feb 81

Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.
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PMID:Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli. 191 Feb 89

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis. 191 67

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli. The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.
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PMID:Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud-induced carboxysomeless mutants. 793 32

The gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activase (rca) was uniformly localized downstream from the genes encoding the large and small subunits of RubisCO (rbcL and rbcS) in three strains of Anabaena species. However, two open reading frames (ORF1 and ORF2), situated between rbcS and rca in Anabaena sp. strain CA, were not found in the intergenic region of Anabaena variabilis and Anabaena sp. strain PCC 7120. During autotrophic growth of Anabaena cells, rca and rbc transcripts accumulated in the light and diminished in the dark; light-dependent expression of these genes was not affected by the nitrogen source and the concentration of exogenous CO2 supplied to the cells. When grown on fructose, rca- and rbc-specific transcripts accumulated in A. variabilis regardless of whether the cells were illuminated. Transcript levels, however, were much lower in dark-grown heterotrophic cultures than in photoheterotrophic cultures. In photoheterotrophic cultures, the expression of the rca and rbc genes was similar to that in cultures grown with CO2 as the sole source of carbon. Although the rbcL-rbcS and rca genes are linked and are in the same transcriptional orientation in Anabaena strains, hybridization of rbc and rca to distinct transcripts suggested that these genes are not cotranscribed, consistent with the results of primer extension and secondary structure analysis of the nucleotide sequence. Transcription from ORF1 and ORF2 was not detected under the conditions examined, and the function of these putative genes remains unknown.
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PMID:Transcription control of ribulose bisphosphate carboxylase/oxygenase activase and adjacent genes in Anabaena species. 796 23

The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the kcat of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219). During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of beta elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits. Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits. The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stablization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits. The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O2). When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to beta eliminate inorganic phosphate from and to misprotonate the enediol intermediate. With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P2 carboxylase's use of this reactive species as a catalytic intermediate.
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PMID:Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits. 903 45

An inactivation library was used to isolate high-CO2-requiring mutants of Synechococcus PCC 7942. One of them, mutant IL-7, is composed of elongated cells, some 5-15 times longer than the wild-type. IL-7 is impaired in the ability to accumulate inorganic carbon within the cells due to a lesion in HCO3- transport. Consequently, the apparent photosynthetic affinity for external inorganic carbon was about 50-100-fold lower than in the wild-type. Analysis of the genomic region modified in IL-7 demonstrated that the inactivating fragment was composed of two genomically unrelated fragments which were ligated together during the formation of the inactivation library. One of the fragments originated from a known genomic region, rbcLS, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase and the other showed high homology to mutS encoding a DNA mismatch repair protein. We suggest that the primary lesion in IL-7 was in mutS and not in rbcLS, and that the phenotype of IL-7 resulted from secondary random mutations. We were unable to identify the spontaneous mutation(s) due to low transformability of IL-7. Our finding that two unrelated fragments ligated together points to possible mistakes in the identification of the function of putative genes with the aid of an inactivation library.
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PMID:A mutant of Synechococcus PCC 7942 impaired in HCO3- uptake. 950 27

Using differential display, we identified the Anabaena sp. PCC 7120 ribulose 1,5-bisphosphate carboxylase transcriptional regulator (rbcR1) gene, a member of the LysR family of positive transcription factors. The rbcR1 transcript and its putative target gene ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL/S) were repressed by cold (20 degrees C) and osmotic (sucrose and salt) stress. Cold stress also induced a transient downregulation of the Anabaena 7120 ntcA transcriptional regulator. Expression of the ntcA gene, however, returned to normal levels 2 h after initiation of cold stress and increased significantly above normal levels 24 h after growth at 20 degrees C. The early decline in the expression of the ntcA, rbcR1, and rbcL/S transcripts appears to be part of the Anabaena 7120 global adaptation response to stress. The substantial increase in the ntcA gene expression 24 h following cold stress suggests that Anabaena 7120 experiences substantial nitrogen limitation under these conditions. These data suggest that in response to stress, Anabaena 7120 decreases its metabolic activity through regulation of the CO(2) fixation machinery while enhancing its nitrogen assimilation by inducing the expression of the nitrogen global transcriptional regulator, NtcA.
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PMID:Transcript levels of rbcR1, ntcA, and rbcL/S genes in cyanobacterium Anabaena sp. PCC 7120 are downregulated in response to cold and osmotic stress. 1216 33


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