Structure-activity analysis of enolpyruvyl transferases

描述(申请人提供)：Mura和EPSPS是烯醇式丙酮基转移酶家族中仅有的已知成员。它们是开发新抗生素药物的有吸引力的目标。穆拉反应是细菌细胞壁生物合成的第一步。EPSP合成酶是莽草酸合成芳香族氨基酸和其他芳香族化合物途径的第六个酶，在许多微生物和植物中都是如此。 这两条途径在哺乳动物中是不存在的，但对微生物的生长是必不可少的。该项目的中心假设是，使用选定的Mura和EPSPS突变酶以及底物、产物和中间态类似物，将允许完整地理解从游离酶到酶产物复合体的整个烯醇式丙酮基转移反应路径。拟议研究的基本原理是，一旦确定了酶反应的先决条件，这些信息最终将被用于设计选择性地针对这两种酶的有效抑制剂。 为了验证我们的假设和实现这个项目的目标，我们将结合蛋白质结晶学、酶动力学、荧光光谱、分子克隆和合成有机化学等技术，追求两个特定的目标：(I)确定烯醇式丙酮酸转移催化的先决条件；(Ii)鉴定诱导Fit机制的先决条件。 这些研究将有助于我们理解烯醇式丙酮酸转移酶的结构和活性之间的关系。此外，它们还将为针对这些重要酶的新型广谱抗菌药物的合理设计提供坚实的基础。

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.