Biosynthesis of Microbial Isoprenoids

描述(申请人提供)：将使用多种化学、生化、结构生物学和生物信息学工具来研究萜类化合物生物合成的生物合成、机械酶学和分子遗传学。重点将放在微生物倍半萜(十五碳萜类)合成酶家族上，这些合成酶催化通用前体法尼基二磷酸环化为大量环倍半萜，包括戊二烯、杰马卡二烯和新发现的化合物表异构烯，以及这些化合物代谢转化为代谢产物，如抗生素戊烯内酯或挥发性有机土豆蛋白，链霉菌和蓝藻的气味成分，导致新鲜翻耕土壤特有的泥土气味，以及与受污染的饮用水和食品相关的不良气味。我们还将挖掘微生物基因组数据库，以鉴定、生化表征和研究细菌和真菌来源的新的萜类合成酶和下游生物合成酶的机制。为了尽可能广泛地探索萜类化合物的机制和结构空间，我们设计了一套相互补充的实验方法，涉及：a.萜类合成酶的机制研究，以确定从FPP形成单个倍半萜的机制。这些研究将包括萜类合成酶的定点突变，利用这种突变体产生异常产物混合物的趋势，这些异常产物是碳阳离子环化级联反应中通常神秘的中间产物的诊断。同位素敏感的支化将被用来建立键的形成和断裂的顺序，并阐明关键的碳阳离子中间体的性质。我们的研究将解决萜类生物合成中的三个核心问题：1)环酶如何在其非环底物FPP及其衍生的中间体上施加特定的构象？2)环酶如何管理正电中间体，包括通过稳定阳离子中间体来催化底物的初始电离，直到通过正电荷猝灭来终止反应？以及3)萜烯合酶序列、结构和功能之间的关系是什么？B.从细菌和真菌来源分离、表达和研究新的萜类合成酶以及来自细菌和真菌的下游生物合成酶，通过基因组序列的生物信息学分析鉴定。C.与David W.Christian教授(宾夕法尼亚大学)合作，测定野生型和突变型萜类合成酶的X射线晶体结构，包括无底物和结合底物和中间类似物，以及与Gerwald Jogl教授(布朗大学)合作，测定选定的下游生物合成酶的结构。 相关萜类代谢物是已知的最大类天然产物，包括激素、抗生素、抗肿瘤药物、免疫抑制剂、神经毒素、风味和气味成分以及色素，以及丰富的医学、生理或商业上的重要性质。研究细菌和真菌对这些化合物的生物合成不仅有助于了解基本的代谢途径，而且有助于开发实用工具，用于产生新的药剂或防止病原微生物或环境微生物形成有毒或其他不良代谢物。最后，利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核心问题--蛋白质序列、结构和功能之间的关系--的基本的新的生物学见解。

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.