Structure-activity analysis of enolpyruvyl transferases

烯醇丙酮酰转移酶的结构-活性分析

• 批准号: 6923008
• 负责人: ERNST SCHONBRUNN
• 金额: $ 25.92万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923008
• 关键词: antiinfective agents; catalyst; chemical transfer reaction; conformation; crystallization; drug design /synthesis /production; enzyme activity; enzyme complex; enzyme inhibitors; enzyme structure; enzyme substrate; fluorescence spectrometry; molecular cloning; phosphoenolpyruvate; protein structure function; site directed mutagenesis; transferase

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.

描述（由申请人提供）：MurA和EPSPS是烯醇丙酮酰转移酶家族中唯一已知的成员。它们是开发新抗生素药物的有吸引力的目标。 MurA 反应是细菌细胞壁生物合成的第一个关键步骤。 EPSP合酶是在许多微生物和植物中合成芳香族氨基酸和其他芳香族化合物的莽草酸途径中的第六种酶。 哺乳动物不存在这两种途径，但对于微生物生长至关重要。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能够全面了解烯醇丙酮酰转移的整个反应途径，从游离酶到酶产品复杂。拟议研究的基本原理是，一旦确定了酶促反应的先决条件，该信息将最终用于设计选择性靶向这两种酶的有效抑制剂。 为了检验我们的假设并实现该项目的目标，将追求两个具体目标，其中整合了蛋白质晶体学、酶动力学、荧光光谱、分子克隆和有机合成化学等技术：（i）鉴定催化烯醇丙酮酰转移的先决条件，以及（ii）诱导拟合机制的先决条件的识别。 这些研究将极大地有助于我们理解烯醇丙酮酰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。