Romana L K, Santiago F S, Reeves P R
Department of Microbiology, University of Sydney, N.S.W., Australia.
Biochem Biophys Res Commun. 1991 Jan 31;174(2):846-52. doi: 10.1016/0006-291x(91)91495-x.
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.
利用T7 RNA聚合酶/启动子系统克隆并过表达了鼠伤寒沙门氏菌LT2的rfbB基因（胸苷二磷酸-D-葡萄糖-4,6-脱水酶）。表达的蛋白占细胞总蛋白的近10%，纯化了14倍。胸苷二磷酸-D-葡萄糖4,6-脱水酶是由43 kDa亚基组成的同型二聚体，对胸苷二磷酸-D-葡萄糖具有高度特异性，对胸苷二磷酸-D-葡萄糖的Km为427 microM，Vmax为0.93微摩尔·分钟-1·微克-1蛋白。N端分析确定了基因在DNA序列中的起始位置。加入对氯汞苯磺酸后酶完全失活，加入巯基乙醇、辅因子NAD+和半胱氨酸后完全重新激活，表明半胱氨酸的一个-SH基团参与催化位点。
