Mechanistic studies of enzymes in isoprenoid biosynthesis

类异戊二烯生物合成酶的机理研究

• 批准号: 8132559
• 负责人: Pinghua Liu
• 金额: $ 31.57万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8132559
• 关键词: Achievement; Address; Affinity Chromatography; Anabolism; Animals; Antibiotics; Artemisinins; Biological Factors; Biomedical Engineering; Biotechnology; Carbon Dioxide; Carotenoids; Chlorophyll; Chloroplasts; Data; Dehydration; Development; Diphosphates; Drug resistance; Electrochemistry; Electron Spin Resonance Spectroscopy; Enzymes; Eubacterium; Foundations; Freezing; Funding; Green Algae; Growth; Health Benefit; Herbicides; Iron; Isomerism; Label; Laboratories; Literature; Malaria; Methods; Mevalonic Acid; Modeling; Molecular Biology; Mossbauer Spectroscopy; NADP; Pathway interactions; Pharmaceutical Preparations; Plants; Play; Precipitation; Production; Proteins; Protocols documentation; Public Health; Reaction; Reagent; Relative (related person); Reporter; Reporting; Rest; Role; Screening procedure; Structure; Sulfur; System; Vascular Plant; Work; analog; artemisinine; base; deoxyxylulose phosphate; enzyme mechanism; enzyme pathway; enzyme substrate; fungus; genome-wide; improved; inhibitor/antagonist; innovation; interest; isopentenyl pyrophosphate; isoprenoid; monomer; mutant; pathogen; protein complex; public health relevance; reconstitution; resistant strain; response; yeast two hybrid system

DESCRIPTION (provided by applicant): All isoprenoids are constructed by isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). The essential role played by the deoxyxylulose phosphate (DXP) pathway in prokaryotic IPP and DMAPP biosynthesis and the lack of this pathway in animals makes the DXP pathway enzymes ideal candidates for developing broad-spectrum antibiotics. The DXP pathway is also targeted for herbicide development because its plant mutants are not able to synthesize sufficient amounts of carotenoids and chlorophylls for normal growth. The low natural abundance of isoprenoids has also stimulated interest in their production through bioengineering. Because the limiting factor in bioengineering-based isoprenoid production is the inadequate supply of IPP and DMAPP, mechanistic studies of the DXP pathway will guide the construction of host strains for bioengineering-based isoprenoid production. The proposed project will study the reaction mechanism of one of the DXP pathway rate-limiting steps, a reductive dehydration reaction catalyzed by an iron-sulfur cluster containing IspH protein. Several major achievements accomplished in preliminary studies serve as the basis for the proposed work. In the preliminary studies, IspH activity was improved by 97-fold relative to that reported in the literature. In addition, using substrate analogs, several IspH mechanistic options were examined and narrowed down to a model that is consistent with all current data. [57Fe]-labeled IspH was isolated in large quantities (~500 mg from a one-day purification). Initial EPR and Mvssbauer characterizations demonstrated that the IspH protein developed has both a high degree of iron- sulfur cluster load and homogeneity. Based on these achievements, the team has acquired all the necessary materials and protocols for conducting the proposed studies. Specifically: In Aim 1, both enzyme- and substrate-based intermediates will be trapped and characterized using a combination of bioorganic and biophysical methods. Several lines of evidence indicate that IspH exists as protein complexes. In Aim 2, by making use of the strains, reagents, and reporter systems obtained in the preliminary studies, several complementary approaches will be utilized to identify IspH partner proteins and study their functions. PUBLIC HEALTH RELEVANCE: The proposed isoprenoid biosynthetic studies will guide the development of mechanism- based inhibitors of the DXP pathway enzymes, which can be used as broad-spectrum antibiotics. The public health benefit will result from the development of effective new treatments for drug-resistant strains of pathogens, currently of increasing concern worldwide.

描述（由申请人提供）：所有类异戊二烯均由异戊烯基二磷酸（IPP）及其异构体二甲基烯丙基二磷酸（DMAPP）构成。磷酸脱氧木酮糖（DXP）途径在原核IPP和DMAPP生物合成中所起的重要作用以及动物中该途径的缺乏使得DXP途径酶成为开发广谱抗生素的理想候选者。DXP途径也是除草剂开发的目标，因为其植物突变体不能合成足够量的类胡萝卜素和叶绿素用于正常生长。类异戊二烯的低天然丰度也激发了通过生物工程生产它们的兴趣。由于生物工程类异戊二烯生产的限制因素是IPP和DMAPP的供应不足，DXP途径的机制研究将指导生物工程类异戊二烯生产宿主菌的构建。拟议的项目将研究DXP途径限速步骤之一的反应机制，即由含有IspH蛋白的铁硫簇催化的还原脱水反应。初步研究中取得的若干重大成果可作为拟议工作的基础。在初步研究中，IspH活性相对于文献中报道的提高了97倍。此外，使用底物类似物，检查了几种IspH机制选项，并缩小到与所有当前数据一致的模型。大量分离[57 Fe]-标记的IspH（一天纯化约500 mg）。初始EPR和Mvssbauer表征表明，所开发的IspH蛋白具有高度的铁-硫簇负载和均一性。在这些成就的基础上，该小组获得了进行拟议研究所需的所有材料和方案。具体而言：在目标1中，将使用生物有机和生物物理方法的组合捕获和表征基于酶和底物的中间体。几条证据表明，IspH作为蛋白质复合物存在。目的二是利用前期研究中获得的菌株、试剂和报告系统，利用多种互补方法鉴定IspH伴侣蛋白并研究其功能。 公共卫生相关性：所提出的类异戊二烯生物合成研究将指导DXP途径酶的基于机制的抑制剂的开发，其可用作广谱抗生素。公共卫生的好处将来自对耐药病原体菌株的有效新疗法的开发，目前在世界范围内日益受到关注。