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 thymidylyltransferase, 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 IjhA 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 , values for dTTP and a-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K , 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 a-D-glucosamine 1-phosphate is accepted equally as well as a-D-glucose 1-phosphate.The NH,-terminal sequence of glucose-1 -phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the 0lf6.l gene of the rj& 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 0rf6.l gene (32453 Da).Saccharides are important surface structures in eukaryotic and prokaryotic cells and involved in cell recognition phenomena. They occur as free saccharides and as glycoconjugates linked to proteins and lipids. One of the problems in studies of their activities is the difficulty in obtaining them in pure form in sufficient quantities. In recent years advances have been made in the chemical synthesis of saccharides [l]. However, saccharide synthesis is still a relatively complicated procedure, in particular when larger saccharides or large quantities are desirable. In the last few years we have been investigating the feasibility of in vitro enzymatic synthesis of the Salmonella enterica 0-antigen-specific oligosaccharide and the nucleotide sugar precursors using enzymes overexpressed by cloned genes [Z].The 0-antigen part of S. enterica sero-group B strains is a repeating tetramer oligosaccharide composed of abequose, mannose, rhamnose and galactose. This saccharide is assembled from appropriate nucleoside &phosphate monosaccharides. The enzymes which participate in the biosynthesis of S. enterica 0-antigen polysaccharide are encoded by genes Abbreviations. Buffer A, 50mM Tris/HCl pH7.6, 10mM MgCl, and 1 mM EDTA; buffer B, 20 mM Tris/HC pH 8.0, 1 mM MgC1, and 22% glycerol; buffer C, 50mM sodium phosphate pH 7.0, 1 M ammonium sulphate and 22% glycerol.Enzymes. Glucose-I-phosphate thymidylyltransferase (EC 2.7.7.24), inorganic pyrophoshatase (EC 3.6.1.1).which are located mostly in the rj8 gene cluster [3]. The rj& gene cluster has been cloned [4-61, and Escherichia coli K12 strains harboring plasmids containing different parts of the rj8 gene cluster of S. enten'ca LT2 was the source of the enzymes used for in vitro enzymatic synthesis of dTDP-Lrhamnose, ...

Conversion of dTDP-4-keto-6-deoxyglucose to free dTDP-4-keto-rhamnose by the rmlC gene products of Escherichia coli and Mycobacterium tuberculosis

“…The genes encoding the first two enzymes in the pathway, rmfA (a-D-glucose-1-phosphate thymidylyltransferase) and rmlB (dTDP-~-glucose-4',6'-dehydratase) have been cloned and expressed from several different organisms (Ma et al, 1997;Lindquist et al, 1993;Romana et af., 1991;Marumo et al, 1992) and shown to catalyse the expected reactions shown in Fig. 1.…”

A Novel NDP-6-deoxyhexosyl-4-ulose Reductase in the Pathway for the Synthesis of Thymidine Diphosphate-d-fucose

“…The synthesis of thymidine diphosphate (dTDP) 1 -L-rhamnose, which is the activated nucleotide sugar form of L-rhamnose, has been well studied by several investigators (15)(16)(17)(18). The anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate is catalyzed by four enzymes, namely, D-glucose-1-phosphate thymidylyltransferase, dTDP-D-glucose 4,6-dehydratase, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and dTDP-4-keto-L-rhamnose reductase ( Fig.…”

“…The anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate is catalyzed by four enzymes, namely, D-glucose-1-phosphate thymidylyltransferase, dTDP-D-glucose 4,6-dehydratase, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and dTDP-4-keto-L-rhamnose reductase ( Fig. 1) (15)(16)(17)(18). On the other hand, D-fucose has been identified as a component of other bacterial polysaccharides in only a few publications (19,20), and there is no report concerning the activated nucleotide sugar form of D-fucose in the synthesis of dTDP-D-fucose.…”

In vitro Synthesis of CDP‐D‐Abequose Using Salmonella Enzymes of Cloned rfb Genes

“…HPLC on SAX anion-exchange columns have earlier been successfully used for assays of the enzymes coded by the Salmonella genes r -A [28], @CD [lo] and r@F [20], and have also been used in studies on the biosynthesis of CDP-D-abequose from CDP-D-glucose, but the CDP-sugars were not resolved under the conditions used [26]. This is the first time, to our knowledge, that CDP-D-abequose has been synthesised vitro in quantitatively significant amounts, and that product as well as the intermediates have been entirely identified.…”