The "green" form I ribulose 1,5-bisphosphate carboxylase/oxygenase from the nonsulfur purple bacterium Rhodobacter capsulatus.

Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified the R. capsulatus form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes from R. capsulatus and R. sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity.

Closely related form I ribulose bisphosphate carboxylase/oxygenase molecules that possess different CO2/O2 substrate specificities.

The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco.

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.