High level expression and purification of dthymidine diphospho-D-glucose 4,6-dehydratase (rfbB) from Salmonella serovar typhimurium LT2.

The rfbB gene (dThymidine-diphospho-D-glucose-4,6-dehydratase) from Salmonella serovar typhimurium LT2 was cloned and over-expressed using the T7 RNA polymerase/promoter system. The expressed protein, which represents almost 10% of the total cellular protein was purified 14-fold. dTDP-D-glucose 4,6-dehydratase is a homodimer of 43 kDa subunits, is highly specific for dTDP-D-glucose and shows a Km of 427 microM and Vmax of 0.93 mu moles min-1 micrograms-1 of protein for dTDP-D-glucose. The N-terminal analysis confirmed the start position of the gene in the DNA sequence. Complete deactivation of the enzyme by the addition of p-chloromercurisulfonic acid and total reactivation by the addition of mercaptoethanol, co-factor NAD+ and cystein showed that a -SH group of the cysteine is involved in the catalytic site.