Grzegorzewicz Anna E, Ma Yufang, Jones Victoria, Crick Dean, Liav Avraham, McNeil Michael R
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Dalian Medical University, Dalian, PR China.
Microbiology (Reading). 2008 Dec;154(Pt 12):3724-3730. doi: 10.1099/mic.0.2008/023366-0.
In Mycobacterium tuberculosis a rhamnosyltransferase (WbbL) catalyses the transfer of an alpha-L-Rhap residue from dTDP-L-rhamnose (dTDP-Rha) to decaprenyldiphosphoryl-alpha-D-N-acetylglucosamine (GlcNAc-P-P-DP) to form alpha-L-Rhap-(1-->3)-alpha-D-GlcNAc-P-P-DP, which is then further elongated with Galf and Araf units, and finally mycolylated and attached to the peptidoglycan. This enzyme is essential for M. tuberculosis viability and at the same time absent in eukaryotic cells, and is therefore a good target for the development of new antituberculosis therapeutics. Here, we report a microtitre plate-based method for the assay of this enzyme using a crude membrane preparation from an Escherichia coli strain overexpressing wbbL as an enzyme source and the natural acceptor substrate GlcNAc-P-P-DP. Initial characterization of the enzyme included unequivocal identification of the product Rha-GlcNAc-P-P-DP by liquid chromatography (LC)-MS, and the facts that WbbL shows an absolute requirement for divalent cations and that its activity is stimulated by beta-mercaptoethanol. Its pH optimum and basic kinetic parameters were also determined, and the kinetic analysis showed that WbbL uses a ternary complex mechanism. The microtitre plate-based assay for this enzyme was developed by taking advantage of the lipophilic nature of the product. This assay should be readily transferable to other glycosyltransferases which use lipid-based acceptors and aid greatly in obtaining inhibitors of such glycosyltransferases for new drug development.
在结核分枝杆菌中，一种鼠李糖基转移酶（WbbL）催化将α-L-鼠李糖残基从二磷酸胸苷-L-鼠李糖（dTDP-鼠李糖）转移至癸二烯基二磷酸-α-D-N-乙酰葡糖胺（GlcNAc-P-P-DP），形成α-L-鼠李糖-(1→3)-α-D- GlcNAc-P-P-DP，然后该产物再进一步与Galf和Araf单元延长，最终进行分枝菌酸化并连接到肽聚糖上。这种酶对于结核分枝杆菌的生存能力至关重要，同时在真核细胞中不存在，因此是开发新型抗结核治疗药物的良好靶点。在此，我们报告了一种基于微量滴定板的方法，用于测定这种酶，该方法使用来自过表达wbbL的大肠杆菌菌株的粗膜制剂作为酶源以及天然受体底物GlcNAc-P-P-DP。该酶的初步表征包括通过液相色谱（LC）-质谱明确鉴定产物鼠李糖-GlcNAc-P-P-DP，以及WbbL对二价阳离子有绝对需求且其活性受β-巯基乙醇刺激这一事实。还确定了其最适pH值和基本动力学参数，动力学分析表明WbbL采用三元复合物机制。基于微量滴定板的该酶测定方法是利用产物的亲脂性开发的。这种测定方法应易于转移到其他使用基于脂质受体的糖基转移酶，并极大地有助于获得此类糖基转移酶的抑制剂以用于新药开发。
