Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.
The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.
Hernandez
J M
JM
Ohio State Biochemistry Program, Ohio State University, Columbus 43210-1292, USA.
Baker
S H
SH
Lorbach
S C
SC
Shively
J M
JM
Tabita
F R
FR
D000595:Amino Acid Sequence; D001483:Base Sequence; D004789:Enzyme Activation; D005798:Genes, Bacterial / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013855:Thiobacillus / Q000201:enzymology* / Q000235:genetics
D012274:Ribulosephosphates; C001933:ribulose-1,5 diphosphate; D012273:Ribulose-Bisphosphate Carboxylase