PURIFICATION, CHARACTERIZATION, AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ASSAY OF SALMONELLA GLUCOSE-1-PHOSPHATE CYTIDYLYLTRANSFERASE FROM THE CLONED RFBF GENE

We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynth esis of CDP-D-abequose from Salmonella enterica strain LT2. The purifi cation was greatly facilitated by using a cloned rfbF gene encoding th is enzyme. Pure enzyme was obtained by 64-fold enrichment in three chr omatography steps. The NH2-terminal sequence of the purified enzyme wa s in agreement with the sequence predicted from the nucleotide sequenc e of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimat ed subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculat ed from the amino acid composition deduced from the nucleotide sequenc e of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catal yzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a ''ping-pong'' type of mechanism. The K(m) values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.