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.
Romana
L K
LK
Department of Microbiology, University of Sydney, N.S.W., Australia.
Santiago
F S
FS
Reeves
P R
PR
D002852:Chromatography, Ion Exchange; D015870:Gene Expression; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D005810:Multigene Family; D010957:Plasmids; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000145:classification / Q000201:enzymology / Q000235:genetics*
D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase