Biosynthesis of Microbial Isoprenoids

微生物类异戊二烯的生物合成

• 批准号: 7373914
• 负责人: DAVID E CANE
• 金额: $ 44.59万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-01-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373914
• 关键词: Address; Amino Acid Sequence; Anabolism; Antibiotics; Bacteria; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Factors; Carbon; Catalysis; Characteristics; Charge; Chemicals; Collaborations; Cyanobacterium; Cyclization; Development; Diagnostic; Enzymatic Biochemistry; Enzymes; Family; Flavoring; Food; Generations; Genes; Genome; Genomics; Hormones; Individual; Investigation; Lead; Metabolic; Metabolic Pathway; Mining; Molecular; Molecular Conformation; Molecular Genetics; Nature; Neurotoxins; Numbers; Object Attachment; Odors; Pennsylvania; Peptide Sequence Determination; Pigments; Prevention; Property; Reaction; Roentgen Rays; Sesquiterpenes; Site-Directed Mutagenesis; Smell Perception; Soil; Source; Streptomyces; Structure; Taste Perception; Terpenes; Terpenoid Biosynthesis Pathway; Universities; analog; antitumor agent; arenaemycin E; drinking water; farnesyl pyrophosphate; fungus; genome database; geosmin; insight; ionization; isoprenoid; microbial; microbial genome; microorganism; mutant; pentalenene; terpene synthase; tool

DESCRIPTION (provided by applicant): The biosynthesis, mechanistic enzymology and molecular genetics of terpenoid biosynthesis will be investigated using a wide variety of chemical, biochemical, structural biological, and bioinformatic tools. The focus will be on a family of microbial sesquiterpene (fifteen-carbon terpenoid) synthases that catalyze the cyclization of the universal precursor farnesyl diphosphate to a large number of cyclic sesquiterpenes, including pentalenene, germacradienol, and the newly discovered compound epi-isozizaene, as well as the metabolic conversion of these compounds to metabolites such as the antibiotic pentalenolactone or the volatile organic geosmin, the odoriferous constituent of Streptomyces and of blue-green algae that is responsible for the characteristic earthy smell of freshly turned soil as well as the undesirable off-taste associated with contaminated drinking water and foods. We will also mine microbial genome databases for identification, biochemical characterization, and mechanistic investigation of new terpenoid synthases and downstream biosynthetic enzymes from bacterial and fungal sources. In order to explore terpenoid mechanism and structure space as broadly as possible, we have devised a set of mutually complementary experimental approaches involving: A. Mechanistic studies of terpene synthases to define the mechanism of formation of individual sesquiterpenes from FPP. These studies will include site-directed mutagenesis of terpene synthases, exploiting the tendency of such mutants to produce mixtures of aberrant products that are diagnostic of the normally cryptic intermediates of the carbocationic cyclization cascade. Isotopically sensitive branching will be used to establish the order in which bonds are made and broken and elucidate the nature of key carbocationic intermediates. Our studies will address three central issues in terpene biosynthesis: 1) How does a cyclase impose a specific conformation on its acyclic substrate FPP and derived intermediates? 2) How does a cyclase manage positively charged intermediates, including catalysis of the initial ionization of the substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? and 3) What are the relationships among terpene synthase sequence, structure, and function? B. Isolation, expression and mechanistic investigation of new terpenoid synthases as well as downstream biosynthetic enzymes from bacterial and fungal sources, identified by bioinformatic analysis of genomic sequences. C. Determination of the X-ray crystallographic structures of wild-type and mutant terpenoid synthases, both substrate-free and with bound substrate and intermediate analogs, in collaboration with Prof. David W. Christianson (University of Pennsylvania), as well as determination of the structures of selected downstream biosynthetic enzymes in collaboration with Prof. Gerwald Jogl (Brown University.) Relevance Terpenoid metabolites, the largest known group of natural products, include hormones, antibiotics, anti- tumor agents, immunosuppresants, neurotoxins, flavor and odor constituents, and pigments, among a wealth of medicinally, physiologically, or commercially important properties. Studies of the biosynthesis of these compounds by bacteria and fungi can not only lead to an understanding of fundamental metabolic pathways, but facilitate the development of practical tools for the generation of new medicinal agents or for prevention of the formation of toxic or otherwise undesirable metabolites by pathogenic or environmental microorganisms. Finally, the use of genome mining to biochemically characterize genes of unknown function will lead to fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function.

描述（由申请人提供）：将使用各种化学、生物化学、结构生物学和生物信息学工具研究萜类化合物生物合成的生物合成、机制酶学和分子遗传学。重点将放在微生物倍半萜家族上（十五碳萜类化合物）脱氢酶，其催化通用前体法呢基二磷酸环化为大量环状倍半萜，包括并环戊二烯、大根香叶烯醇和新发现的化合物表异齐烯，以及这些化合物代谢转化为代谢物，如抗生素并环戊二烯内酯或挥发性有机土臭素，链霉菌属和蓝绿藻的有气味的成分，造成新翻的泥土特有的泥土气味以及与受污染的饮用水和食物有关的令人不快的味道。我们还将挖掘微生物基因组数据库，用于鉴定，生物化学表征和新的萜类化合物脱氢酶和来自细菌和真菌来源的下游生物合成酶的机理研究。为了尽可能广泛地探索萜类化合物的作用机制和结构空间，我们设计了一套相互补充的实验方法。萜烯脱氢酶的机制研究，以确定从FPP形成单个倍半萜烯的机制。这些研究将包括萜烯脱氢酶的定点诱变，利用这种突变体产生异常产物的混合物的趋势，这些异常产物是碳阳离子环化级联的通常隐蔽的中间体的诊断。同位素敏感性支化将用于建立键的形成和断裂顺序，并阐明关键碳阳离子中间体的性质。我们的研究将解决萜烯生物合成中的三个中心问题：1）环化酶如何将其非环底物FPP及其衍生中间体强加于特定构象？2)环化酶如何管理带正电荷的中间体，包括催化底物的初始电离，通过稳定阳离子中间体，通过正电荷的猝灭终止反应？萜类合成酶序列、结构和功能之间的关系是什么？B。通过基因组序列的生物信息学分析鉴定的来自细菌和真菌来源的新的萜类化合物脱氢酶以及下游生物合成酶的分离、表达和机理研究。C.与大卫W教授合作，测定野生型和突变型萜类化合物脱氢酶的X射线晶体学结构，包括无底物和有结合底物和中间类似物。Christianson（宾夕法尼亚大学），以及与Gerwald Jogl教授（布朗大学）合作确定选定的下游生物合成酶的结构。 相关性萜类代谢物是已知最大的天然产物组，包括激素、抗生素、抗肿瘤剂、免疫抑制剂、神经毒素、风味和气味成分以及色素，它们具有丰富的医学、生理学或商业上重要的性质.通过细菌和真菌对这些化合物的生物合成的研究不仅可以导致对基本代谢途径的理解，而且可以促进用于产生新的药物或用于防止致病或环境微生物形成有毒或其他不期望的代谢物的实用工具的开发。最后，使用基因组挖掘对未知功能的基因进行生物化学表征，将导致对现代分子生物化学的中心问题，蛋白质序列，结构和功能之间的关系的基本新的生物学见解。