Enzymatic synthesis and isolation of thymidine diphosphate‐6‐deoxy‐D‐xylo‐4‐hexulose and thymidine diphosphate‐L‐rhamnose

Abstract

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rjb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-~-xylo-4-hexulose was isolated after a 1 -h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89Y0). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-~-xylo-4-hexulose 3,5-epimerase and NADPH : dTDP-6-deoxy-~-l.vxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was ~W O , corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-~-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-~-.xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier.Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-~-xy/o-4-hexulose 3,5-epimerase and NADPH : dTDP-6-deoxy-~-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-~-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.The 0 antigen part of the lipopolysaccharide of Salmonella enterica sero-group B strains has a saccharide chain built of site interaction. Therefore, isolated saccharides are important for elucidation of host-defence mechanisms.In recent years advances have been made in the chemical synthesis of saccharides [3]. However, saccharide synthesis is still a relatively complicated procedure, in particular when saccharides larger than the tetrasaccharides shown above are to be synthesized and synthesis is required in large quantities. Such saccharides may be required for the production of synthetic vaccines. S. entericu 0-antigen-specific oligosaccharides have also been produced by enzymatic degradation of the polysaccharide, utilizing bacteriophage glycosidases [4]. So far this procedure has yielded oligosaccharides (monotetramers, ditetramers, tritetramers, and tetramers of a tetrasaccharide) which have terminal sugars different from those of the natural biological repeating unit. We have therefore investigating the feasability of enzymatic synthesis of the S. enterica 0-antigenspecific oligosaccharide using enzymes overproduced by cloned gene...

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

“…ATP, CTP, GTP, ITP, UTP, ADP-D-glucose, CDP-Dglucose, dTDP-D-glucose, UDP-D-glucose, UDP-D-galactose, UDP-D-mannose, UDP-N-acetyl-D-glucosamhe, and all sugar 1-phosphates used were from Sigma Chemical Co. (St Louis, MO, USA). The dTDP-L-rhamnose was prepared as described [2].…”

“…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 enzymes used for in vitro enzymatic synthesis of dTDP-Lrhamnose, starting from dTDP-L-glucose [2].…”

“…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, starting from dTDP-L-glucose [2].…”

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.…”