Structural investigations, at atomic level, of three enzymes comprising steps three, four and five of the mevalonate-independant pathway for isoprenoid biosynthesis

这项提议的目的是了解细菌中异戊二烯及其化学前体生物合成的结构和机制特征。异戊二烯是自然界中发现的结构最多样化的化合物家族。迄今为止，已知的类异戊二烯分子超过23.000个[1]。许多异戊二烯类化合物具有生物技术应用，如药物、香料、颜料、香水或农用化学品。在生物体内，类异戊二烯在多种电子传递、生殖、生长调节、信号转导和防御等过程中发挥着重要作用。我建议使用蛋白质X射线结晶学与功能实验相结合的方式，包括定点突变和稳态动力学分析，以解决在原子分辨率下通过所谓的甲戊酸非依赖或甲基-D-赤藓糖醇(MEP)途径合成类异戊二烯的立体化学和动力学基础[2]。这项研究计划将重点放在与三种酶的动力学受损突变体复合的底物的结构解释上，这三种酶包括MEP生物合成途径的第三步、第四步和第五步。由于MEP途径在人类中不存在，并已被证明在细菌中是必不可少的，其酶是治疗细菌和寄生虫感染的新药靶点的极佳候选者。

The objective of this proposal is to understand the structural and mechanistic features governing the biosynthesis of isoprenoids and their chemical precursors in bacteria. Isoprenoids are the most structurally diverse family of compounds found in nature. More than 23.000 isoprenoid molecules are know to date [1]. Many isoprenoids have biotechnological applications as drugs, flavours, pigments, perfumes or agrochemicals. In living beings, isoprenoids have important roles in processes as diverse electron transport, reproduction, growth regulation, signal transduction and defence. I propose to use protein x-ray crystallography in an integrated fashion with functional experiments including site-directed mutagenesis and steady state kinetic analysis to address the stereochemical and kinetic basis for isoprenoid biosynthesis through the so-called mevalonate-independent or methyl-D-erythritol (MEP) pathway at atomic resolution [2]. This research proposal will focus on the structural elucidation of substrates complexed with kinetically impaired mutants of three enzymes comprising steps three, four, and five of the MEP pathway for isoprenoid biosynthesis. Since the MEP pathway is absent in humans and has been shown to be essential in bacteria, its enzymes are excellent candidates for new drug targets, for the treatment of both bacterial and parasitic infections.