Amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from Euglena

Summary The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10^-9 per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.Abbreviations DABITC Dimethyl amino azobenzene isothiocyanate - HPLC high pressure liquid chromatography - Kd Kilo daltons - LSU large subunit - PITC phenyl isothiocyanate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - SSU small subunit - TFA trifluoric acetic acid     

RNA binding activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit from Chlamydomonas reinhardtii

Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289-16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5'-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.     

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.     

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].     

Activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in three marine microalgae

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in three marine microalgae: the chlorophyte t Dunaliella tertiolecta and the chromophytes t Pavlova lutheri and t Thalassiosira pseudonana. The three species differed in the sensitivity of Rubisco activity in crude extracts to magnesium ion concentration, the presence of protease inhibitors, the duration of the incubation on activity, and the potential for full activation of Rubisco with 20 mM magnesium chloride and 20 mM bicarbonate t in vitro. t D. tertiolecta had responses that were similar to those described in vascular plants: regulation of initial activity on a gradient of irradiances; maximum initial activities that were 80– 90% of light-saturated photosynthesis; total activities that exceeded light-saturated photosynthesis by 30–100%; and deactivation of Rubisco in darkness. Both initial and total activity declined in darkness and increased on a return to growth irradiance. First-order time constants were about 9 min for deactivation and 3 min for reactivation of initial activity. The decline in total activity after a transition into darkness could not be reversed t in vitro but could be reversed by exposing t D. tertiolecta to light, a characteristic of regulation by CA1P. The responses of t T. pseudonana were qualitatively similar, except that recovery of initial activity was low and could only account for 30–40% of light-saturated photosynthesis. Rubisco from t T. pseudonana exposed to low irradiance could be activated t in vitro but at growth irradiance and higher, total activity was lower than initial activity. The time constants for deactivation and reactivation of initial activity after reciprocal switches between growth irradiance and darkness were 12–18 min and 3 min in t T. pseudonana. t P. lutheri showed no regulation of Rubisco activity in response to changes in irradiance or light-dark transitions. This may have been an artifact of the conditions chosen to measure activity.     

Isolation and partial amino acid sequencing of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from three species of orchid

Small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been purified from 3 species of orchid in the genus Cymbidium by gel filtration followed by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. The samples were subjected to amino acid composition analysis and partial N-terminal amino acid sequencing. The sequencing data clearly confirm that one of the species examined is the hybrid offspring of a cross between the other two.     

Complete processing of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea requires the amino acid sequence Ile-Thr-Ser

Chloroplast import and processing of two precursor proteins with mutations in the carboxyl-terminal region of the transit peptide were examined in vitro. Deletion mutations were introduced into the 57-amino acid transit peptide of a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, from pea. A mutant, PSd48/57, in which nine carboxyl-terminal amino acids of the transit peptide had been deleted, was imported and processed to a series of 13- to 18-kDa polypeptides including the 14-kDa mature small subunit. In contrast, processing of a mutant, PSd45/57, in which an additional three amino acids had been removed, resulted in a series of polypeptides which did not include the mature small subunit. Whereas PSd48/57 was imported as efficiently as the wild-type precursor, import of PSd45/57 was only 25% as efficient as that of the authentic precursor. The mutant precursor proteins PSd48/57 and PSd45/57 are distinguished by a three-amino acid sequence, Ile-Thr-Ser, located in the carboxyl-terminal region of the transit peptide. We show that all or part of this sequence is required for correct processing.     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase

The gene for the large subunit (LS) of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBPCase/ Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced arnino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6–10 bp upstream from the initiation codon.     

Physical, immunological and kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase from fir (Abies alba) and spruce (Picea abies)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from fir ( Abies alba Mill.) and spruce ( Picea abies [L.] Karst.) needles was purified to homogeneity. The enzyme was isolated from crude extracts through quantitative precipitation in 40-55% and 40-60% (NH₄)₂SO₄ for fir and spruce. respectively, followed by linear sucrose gradient centrifugation. Using two dimensional gel electrophoresis, the isoelectric points were determined. For the large subunit (LSU) it was 6.7 for both species, and for the small subunit (SSU) it was 7.1 and 7.7 for fir and spruce, respectively. Very few differences in tryptic peptides and amino acid composition of Rubisco LSU were observed between fir and spruce. By contrast, marked differences characterized the same analyses for the Rubisco SSU of the two species. Moreover, substitution of residues was observed in the sequenced N-terminal region when comparing fir and spruce SSU. The Ouchterlony technique showed no immu-nochemical difference between Rubisco of fir and spruce when a rabbit antiserum to spinach Rubisco was used. The Eadie-Hofstee plots of carboxylase activity indicated that the apparent K_m(CO₂) were 31 and 36 μ M for the fir and spruce enzymes, respectively.     

Two different genes and gene products for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCOase) in Nitrobacter hamburgensis

Abstract Heterologous DNA hybridization using a ribulose- 1,5-biphosphate carboxylase/oxygenase (RuBisCOase) large subunit gene ( rbc L) probe from Anacystis nidulans revealed the presence of two rbc L in Nitrobacter hamburgensis . One gene is located on a plasmid, the other on the chromosome. The genes appear to be very similar since both hybridized strongly to the A. nidulans probe. However, restriction endonuclease digestions revealed differences.
Two different RuBisCOase enzymes were isolated from N. hamburgensis. The M _r of the native enzymes were 520 000 and 480 000. Sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) revealed the presence of both LSU and small subunits (SSU) for both enzymes. The M _r were 53 000 and 16 000, and 49 000 and 13 500, respectively. A hexadecameric structure is suggested for both enzymes.     

Truncation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum affects the holoenzyme assembly and activity.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site-directed mutagenesis to examine the role of the C-terminal tail section. Removal of the last and the penultimate alpha-helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild-type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end-to-end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2-carboxy arabinitol-1,5-bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C-terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.     

The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath)

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.