Characterization of glutamine synthetase isoforms from chlorella

Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GS_I and GS_II were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GS_I and 43,000 for GS_II. The amino acid composition, catalytic, and immunological properties for both enzymes are similar.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor.

Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301. The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E. coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes. Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively. Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP. The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate. Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO. Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins. These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.     

Overproduction of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus sp. strain PCC6301 in glucose-controlled high-cell-density fermentations by Escherichia coli K-12.

A predictive and feedback glucose feed controller, previously developed for nutrient-sufficient growth of Escherichia coli to high cell densities, was used to produce large quantities of a heterologous, cyanobacterial recombinant hexadecameric (L8S8) protein, ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in E. coli. Culture and plasmid stability conditions were optimized to yield the production of approximately 1 g of soluble, active recombinant RubisCO per liter. Recombinant RubisCO also was produced in lactose-induced high-cell-density fermentation of E. coli K-12.     

Photolithoautotrophic growth and control of CO2 fixation in Rhodobacter sphaeroides and Rhodospirillum rubrum in the absence of ribulose bisphosphate carboxylase-oxygenase.

Rhodospirillum rubrum and Rhodobacter sphaeroides were shown to be capable of photolithoautotrophic growth in the absence of the reductive pentose phosphate (Calvin) cycle. Ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strains were incapable of photolithoautotrophic growth using hydrogen as an electron donor but were able to grow in the absence of organic carbon using less reduced inorganic electron donors, i.e., thiosulfate or sulfide. Wild-type R. rubrum grown in the presence of thiosulfate contained RubisCO levels that were 50-fold lower compared with those in cells growth with hydrogen as an electron donor without substantially influencing rates of photolithoautotrophic growth. These results suggest there are two independent CO2 fixation pathways that support photolithoautotrophic growth in purple nonsulfur photosynthetic bacteria, indicating that these organisms have developed sophisticated control mechanisms to regulate the flow of carbon from CO2 through these separate pathways.     

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.