Structural Basis for Isoprenoid Biosynthesis

描述：(申请人提供)本研究的长期目标 计划是了解类异戊二烯的结构基础 细菌和寄生虫的生物合成，这将成为 新型抗菌抗疟疾药物的设计。在……里面 特别是，这项建议的一个关键目标是理解结构和 类异戊二烯前体生物合成的机理特征 异戊烯基二磷酸(IPP)及其异构体二甲基烯丙基 甲氧戊酸非依赖性酶催化的二磷酸(DMAPP) 路径。此外，对最近一次的结构和功能分析 机制和结构上不同的新型IPP异构酶 从前面描述的IPP异构酶将被采用。此类型2 IPP 异构酶与研究广泛的1型酶不同，它依赖于黄素 单核苷酸和NADPH活性。值得注意的是，这种类型2的IPP 在几种革兰氏阳性菌中发现异构酶，如葡萄球菌 金星表明它将成为新的 抗葡萄球菌药物。最后，细菌形式的结构 从大肠杆菌中获得的法尼基二磷酸合成酶将被 下定决心。IPP和DMAPP是异戊二烯类化合物的基本构件 所有的有机体，是构建无数 高级萜类化合物。最近的独立研究证明了这种现象的存在 一种新的甲氧戊酸非依赖的IPP合成途径，称为 1-脱氧-D-木糖5-磷酸/2-C-甲基-D-赤藓糖醇4-磷酸(DXP/MEP) 路径。后一种甲氧戊酸非依赖性途径利用丙酮酸和 以3-磷酸甘油醛为原料生产IPP。这个 DXPIMEP途径存在于多种真细菌中，包括几个 致病物种，如结核杆菌，在藻类中，在 恶性疟原虫植物细胞和质外体中的质体 导致疟疾的寄生虫。鉴于DxP/MEP途径的本质 在这些生物体中，以及在哺乳动物中缺乏这一途径时，这些酶 包括DxP/MEP途径代表了产生 选择性抗菌剂和抗疟疾药。为了更好地了解 这条途径的机制特征以及随后形成的 关键的二磷酸盐中间体金合欢二磷酸(FPP)，我们已经开始了 DXP/MEP途径和DXP/MEP途径组成酶的结构解析 IPP异构酶和FPP合酶分别形成DMAPP和FPP， 使用蛋白质X-射线结晶学。

DESCRIPTION: (provided by applicant) The long term objective of this research plan is to gain an understanding of the structural basis for isoprenoid biosynthesis in bacteria and parasites, which will serve as a foundation for the design of new classes of antibacterial and antimalarial agents. In particular, a key aim of this proposal is to understand the structural and mechanistic features governing the biosynthesis of the isoprenoid precursors isopentenyl disphosphate (IPP) and the isomeric compound, dimethylallyl diphosphate (DMAPP) catalyzed by enzymes comprising the mevalonate-independent pathway. In addition, the structural and functional analysis of a recently described and novel IPP isomerase that differs mechanistically and structurally from previously described IPP isomerases will be undertaken. This type 2 IPP isomerase, unlike the well-studied type 1 enzymes, depends on flavin mononucleotide and NADPH for activity. Notably, this type 2 class of IPP isomerase is found in several Gram-positive bacteria such as Staphylococcus aureus suggesting that it will serve as an effective target for new anti-staphylococcal drugs. Finally, the structure of the bacterial form of the enzyme farnesyl diphosphate synthase obtained from Escherichia coli will be determined. IPP and DMAPP are the fundamental building blocks of isoprenoids in all organisms and are essential metabolites for the construction of numerous higher order terpenoids. Recent independent studies demonstrated the existence of a novel, mevalonate-independent pathway for IPP synthesis known as the 1-deoxy-D-xylulose 5-phosphate / 2-C-methyl-D-erythritol 4-phosphate (DXP/MEP) pathway. This latter mevalonate-independent pathway utilizes pyruvate and glyceraldehyde 3-phosphate as starting materials for production of IPP. The DXPIMEP pathway occurs in a variety of eubacteria that includes several pathogenic species such as Mycobacterium tuberculosis, in algae, in the plastids of plant cells and in the apicoplast of Plasmodium falciparum the parasite that causes malaria. Given the essential nature of the DXP/MEP pathway in these organisms and the absence of this pathway in mammals, the enzymes comprising the DXP/MEP pathway represent targets for the generation of selective antibacterial and antimalarial agents. In order to better understand the mechanistic features of this pathway and the subsequent formation of a critical diphosphate intermediate farnesyl diphosphate (FPP), we have begun the structural elucidation of the enzymes comprising the DXP/MEP pathway and the formation of DMAPP and FPP from IPP isomerase and FPP synthase, respectively, using protein x-ray crystallography.