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（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）构建。磷酸脱氧木酮糖 (DXP) 途径在原核 IPP 和 DMAPP 生物合成中发挥重要作用，而动物体内缺乏该途径，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，因为其植物突变体无法合成足够量的类胡萝卜素和叶绿素以维持正常生长。类异戊二烯的天然丰度较低也激发了人们对通过生物工程生产其的兴趣。由于IPP和DMAPP供应不足是基于生物工程的类异戊二烯生产的限制因素，因此DXP途径的机理研究将指导基于生物工程的类异戊二烯生产宿主菌株的构建。该项目将研究 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。初步研究中取得的几项重大成果是拟议工作的基础。在初步研究中，IspH 活性相对于文献报道提高了 97 倍。此外，使用底物类似物，检查了几种 IspH 机械选项，并将范围缩小到与所有当前数据一致的模型。大量分离出[57Fe]标记的 IspH（一天纯化约 500 mg）。最初的 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质性。基于这些成就，该团队已经获得了进行拟议研究所需的所有材料和协议。具体来说：在目标 1 中，将使用生物有机和生物物理方法的组合来捕获和表征基于酶和基于底物的中间体。多项证据表明 IspH 以蛋白质复合物的形式存在。在目标2中，通过利用在初步研究中获得的菌株、试剂和报告系统，将利用几种补充方法来鉴定IspH伙伴蛋白并研究其功能。 公共健康相关性：拟议的类异戊二烯生物合成研究将指导基于机制的 DXP 途径酶抑制剂的开发，该抑制剂可用作广谱抗生素。针对目前全世界日益关注的耐药病原体菌株，开发有效的新疗法将带来公共卫生益处。