PEPTIDOGLYCAN BIOSYNTHESIS AND VANCOMYCIN RESISTANCE

本提案重点研究酶在细菌细胞壁组装中的作用， 肽聚糖（PG）组分，其是细菌特有的结构，并且已知 成为几种临床上有用的抗生素的目标。 实验 在两个方面提出：（1）PG中的第一个承诺步骤 生物合成，从PEP到UDP-N乙酰基的不寻常烯醇式转移 葡糖胺，以产生UDPenolvyl G1 cNAc，支架元件 用于肽组装和（2）形成肽组装的PG的D-ala-D-ala末端。 抗生素万古霉素的高亲和力位点。 我们最近 克隆、测序并纯化至均一MurZ，烯醇化酶 转移酶，并建议研究其催化机制和 抗生素对该酶的时间依赖性失活机制 磷霉素，在欧洲临床使用的环氧丙烷膦酸盐。 没有 已知磷霉素的特异性的分子信息， MurZ和催化机制的结构/功能研究可能导致 针对这一目标改进了抗生素设计。 危及生命的革兰氏阳性菌出现万古霉素耐药性 感染（例如，当货车耐药基因编码五种新的 蛋白质、VanS、R、H、A、X。 我们最近过度生产，纯化， 特征在于VanH，D-特异性α-酮酸还原酶和VanA，D-Ala-丙氨酸还原酶。 D-X连接酶，并表明它们协同作用，使D-丙氨酸-D-乳酸（a 缩肽）并允许正常的D-Ala-D-Ala PG末端的置换 被D-丙氨酸-D-乳酸盐取代，不再识别万古霉素。 我们提出 进一步研究万古霉素耐药的分子机制， VanA与染色体D-Ala-D-Ala连接酶的比较以及 为了纯化和表征VanS和VanR， 用于控制VanH，A，X转录的调节系统。 知识 所述货车抗性蛋白可允许设计药物，例如磷酸化的药物， phinate二肽模拟物，使万古霉素耐药菌 灵敏度

This proposal focuses on enzymes in bacterial cell wall assembly of the peptidoglycan (PG) component, a structure unique to bacteria and known to be the target of several clinically useful antibiotics. Experiments are proposed in two areas: (1) the first committed step in the PG biosynthesis, an unusual enolpyruvyl transfer from PEP to UDP-Nacetyl gluco-samine to produce UDPenolpyruvyl G1cNAc, the scaffolding element for peptide assembly and (2) the D-ala-D-ala termini of PG that form the high affinity site for the antibiotic vancomycin. We have recently cloned, sequenced and purified to homogeneity MurZ, the enolpyruvyl transferase, and propose to study its catalytic mechanism and the mechanism of time-dependent inactivation of this enzyme by the antibiotic fosfomycin, an epoxypropane phosphonate in clinical use in Europe. No molecular information is known about the specificity of fosfomycin for MurZ and structure/function studies on catalytic mechanism could lead to improved antibiotic design against this target. Vancomycin resistance arises in life-threatening gram positive bacterial infections (e.g., endocarditis) when Van resistance genes encode five new proteins, VanS, R, H, A, X. We have recently overproduced, purified and characterized VanH, a D-specific a-ketoacid reductase and VanA, a D-Ala- D-X ligase and shown that they act in concert to make D-Ala-D-Lactate (a depsipeptide) and allow replacement of the normal D-Ala-D-Ala PG terminus by D-Ala-D-Lactate and that no longer recognizes vancomycin. We propose to further study the molecular mechanism of vancomycin resistance by comparison of VanA with the chromosomal D-Ala-D-Ala ligases as well as to purify and characterize VanS and VanR, a proposed two component regulatory system for control of VanH, A, X transcription. Knowledge of the Van resistance proteins may permit design of drugs, such as phos- phinate dipeptidomimetics, to revert vancomycin resistant bacteria to sensitivity.