Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

Abstract

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 Km values for dTTP and α‐d‐glucose 1‐phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the Km 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 α‐d‐glucosamine 1‐phosphate is accepted equally as well as α‐d‐glucose 1‐phosphate. The NH2‐terminal sequence of glucose‐1‐phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the orf6.1 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). Copyright © 1993, Wiley Blackwell. All rights reserved