PURIFICATION, CHARACTERIZATION AND HPLC ASSAY OF SALMONELLA GLUCOSE-1-PHOSPHATE THYMIDYLYTRANSFERASE FROM THE CLONED RFBA GENE

We here report on the purification and characterization of glucose-1-phosphate thymidylyl-transferase, the first of four enzymes commited to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the rfbA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps. The glucose-1-phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a 'ping-pong' mechanism. The K(m) values for dTTP and alpha-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K(m) values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP-D-glucose and alpha-D-glucosamine 1-phosphate is accepted equally as well as alpha-D-glucose I-phosphate. The NH2-terminal sequence of glucose-1-phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the or 6.1 gene of the rfb gene cluster. The SDS/PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene (32453 Da).