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 f orm I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (Rub isCO), respectively, were found to complement a RubisCO-negative mutan t of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. deni trificans promoters were shown to function in R. sphaeroides, resultin g in high levels of cbbL cbbS and cbbM expression in the R. sphaeroide s host. This expression system provided high levels of both T. denitri ficans enzymes, each of which was highly purified. The deduced amino a cid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes fro m sulfur oxidizing bacteria. The form I T. denitrificans enzyme posses sed a very low substrate specificity factor and did not exhibit fallov er, and yet this enzyme showed a poor ability to recover from incubati on with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II R ubisCO enzymes. The substrate specificity factor was characteristicall y low, and the lack of fallover and the inhibition by ribulose 1,5-bis phosphate were similar to those of form II RubisCO obtained from nonsu lfur purple bacteria. Both form I and form II RubisCO from T. denitrif icans possessed high K-CO2 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 f orm I and form II RubisCO from a chemoautotrophic bacterium that synth esizes both types of enzyme.