Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Purification and characterization of maize ribulose-1,5-bisphosphate carboxylase

The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C₄ monocot) to homogeneity has a MW of532 000 and sedimentation coeffici     

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley leaves was purified by Sepharose 6B gel filtration at pH 8.3 as a single, colorless peak containing both activities. Approximately 0.2 g atom copper per mole enzyme was detected by atomic absorption spectroscopy, but this copper was not detectable by EPR spectrometry.     

Amino acid sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase of Pisum sativum

The amino acid sequence of the small subunit of ribulose-1, 5-bisphosphate carboxylase from pea consists of a single polypeptide chain of 123 residues with a calculated MW of ca 14 480. The N-terminus was ‘ragged’ and both methionine and glutamine were determined in residue position 1. No heterogeneity was found even though two isofocussing variants were observed. The amino acid sequence confirms the nucleic acid sequence of cDNA of mRNA determined independently.     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.     

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].     

Disparate distributions of chemolithotrophs containing form IA or IC large subunit genes for ribulose-1,5-bisphosphate carboxylase/oxygenase in intertidal marine and littoral lake sediments

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.     

Taxomomic variation in amino acid compositions of ribulose-1,5-bisphosphate carboxylases from grasses

Ribulose-1,5-biphosphate (RuBP) carboxylases from 38 grass species (26 genera), isolated via affinity chromatography, compare well in amino acid compositions with the enzyme extracted and purified from the same and other species (46 species, 38 genera) by an alternative procedure. Taxonomic differences in the amino acid composition of the enzyme exist between the major grass groups. Those of pooids are distinguishable from those of chloridoids, eu-panicoids and andropogonoids, while those of a bamboo, Oryza, Microlaena and Stipeae bear closer resemblance to those of pooids. The amino acid composition of RuBP carboxylases does not resemble that of total leaf proteins of the grasses. Variations detected in the amino acid compositions of RuBP, carboxylases are independent of observed differences in kinetics between C₃ and C₄ versions of the enzyme.     

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

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.