Structure-activity analysis of enolpyruvyl transferases

描述（由申请人提供）：MurA和EPSPS是烯醇化酰基转移酶家族的唯一已知成员。它们是开发新抗生素药物的有吸引力的靶点。MurA反应形成细菌细胞壁生物合成中的第一个关键步骤。EPSP合成酶是许多微生物和植物中合成芳香族氨基酸和其它芳香族化合物的莽草酸途径中的第六种酶。 这两种途径在哺乳动物中都不存在，但对微生物生长至关重要。所提出的项目的中心假设是，使用选定的突变酶的MurA和EPSPS与底物，产品和中间状态类似物将允许一个完整的了解烯醇化酶转移的整个反应途径，从游离酶的酶产物复合物。这项研究的基本原理是，一旦确定了酶促反应的先决条件，这些信息将最终用于设计选择性靶向两种酶的强效抑制剂。 为了检验我们的假设，并完成本项目的目标，两个具体的目标，其中集成技术，如蛋白质晶体学，酶动力学，荧光光谱，分子克隆，和合成有机化学，将追求：（i）的烯醇式转移的催化的先决条件的识别，和（ii）的诱导适合机制的先决条件的识别。 这些研究将大大有助于我们了解烯醇式丙酮酸转移酶的结构和活性之间的关系。此外，它们将为合理设计针对这些重要酶的新型广谱抗菌药物提供坚实的基础。

DESCRIPTION (provided by applicant): MurA and EPSPS are the only known members of the enolpyruvyl transferase family of enzymes. They are attractive targets for the development of new antibiotic drugs. The MurA reaction forms the first committed step in the biosynthesis of the bacterial cell wall. EPSP synthase is the sixth enzyme in the shikimate pathway towards the synthesis of aromatic amino acids and of other aromatic compounds in numerous microorganisms and plants. Both these pathways are absent from mammals but essential for microbial growth. The central hypothesis to the proposed project is that the use of selected mutant enzymes of MurA and EPSPS together with substrate, product and intermediate state analogs will allow for a complete understanding of the entire reaction pathway of enolpyruvyl transfer, starting from free enzyme to the enzyme products complex. The rationale for the proposed research is that once the prerequisites for the enzymatic reaction are identified, this information will be ultimately utilized to design potent inhibitors that selectively target both enzymes. In order to test our hypothesis and accomplish the objective of this project, two specific aims, which integrate techniques such as protein crystallography, enzyme kinetics, fluorescence spectroscopy, molecular cloning, and synthetic organic chemistry, will be pursued: (i) the identification of the prerequisites for the catalysis of enolpyruvyl transfer, and (ii) the identification of the prerequisites for the induced-fit mechanism. These studies will substantially contribute to our understanding of the relationship between the structure and activity of enolpyruvyl transferases. Moreover, they will provide a solid basis for the rational design of novel broad-spectrum antimicrobial drugs targeting these important enzymes.