Barbosa Maria D F S, Ross Harold O, Hillman Milton C, Meade Raymond P, Kurilla Michael G, Pompliano David L
Department of Antimicrobial Research, Bristol-Myers Squibb Pharmaceuticals, Wilmington, Delaware 19880, USA.
Anal Biochem. 2002 Jul 1;306(1):17-22. doi: 10.1006/abio.2001.5691.
Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG. Conditions have been optimized for the production of large amounts of membranes with increased levels of MurG, allowing the development of an assay suitable for high-throughput screening of large compound libraries. The quality of the purified membranes was assessed by electron microscopy and also by testing cross-linked peptidoglycan production. Moreover, kinetic studies allowed the determination of optimal concentrations of the substrates and membranes to be utilized for maximum sensitivity of the assay. Using a 96-well assay format, the IC50 values for vancomycin, tunicamycin, flavomycin, and bacitracin were 1.1 microM, 0.01 microg/ml, 0.03 microg/ml, and 0.7 microg/ml, respectively.
肽聚糖合成始于细胞质中，由UDP-N-乙酰葡糖胺烯醇丙酮酸转移酶催化UDP-N-乙酰葡糖胺（UDP-GlcNAc）与磷酸烯醇丙酮酸缩合。UDP-GlcNAc还用作糖基转移酶MurG的底物，MurG是一种膜结合酶，催化脂质II的产生。过量表达MurG的大肠杆菌细胞的膜支持肽聚糖形成的速率比含有野生型水平MurG的膜快约五倍。已优化条件以生产大量具有更高MurG水平的膜，从而开发出一种适用于高通量筛选大型化合物库的检测方法。通过电子显微镜以及测试交联肽聚糖的产生来评估纯化膜的质量。此外，动力学研究确定了用于该检测方法最大灵敏度的底物和膜的最佳浓度。使用96孔检测形式，万古霉素、衣霉素、黄霉素和杆菌肽的IC50值分别为1.1微摩尔、0.01微克/毫升、0.
