Biosynthesis of Microbial Isoprenoids

描述(申请人提供)：将研究细菌和真菌中萜类化合物生物合成的生物合成、机制酶学和分子遗传学，特别强调通过挖掘基因组数据库发现的三大类蛋白质的机制和结构特征，以及呈现新的结构、机制或生物学特征的功能基因表达：1)萜烯合成酶的研究将集中于确定环化机制和阐明每种蛋白质在催化形成其单独的环化产物中的作用。生物信息学工具以及突变方法的组合将被用来识别代表新的结构类型或不寻常的生物合成反应和机制的靶标，或者对应于目前未知功能的隐蔽合酶的靶标，这些合酶涉及重要的生物过程，如真菌毒力。机制的重点将是发现新的环酶，并利用定点突变和蛋白质结晶学的组合来解开催化和产物特异性的蛋白质结构基础。2)将研究多种催化新型加氧酶，这些加氧酶在萜类碳氢化合物和醇向生物合成最终产物的代谢转化中发挥关键作用。3)还将探讨微生物萜类生物合成的调控，重点是两个有趣的模型系统，即抗生素戊烯内酯生物合成的调控和探索与广泛存在的挥发性有机代谢物2-甲基异龙脑的生物合成基因相关的高度保守的核苷酸结合蛋白的隐蔽作用。数以万计的萜类天然产物存在于陆地和海洋植物、真菌和苔类植物中，以及大量的细菌中。其中许多化合物被广泛用于人类医学，如抗肿瘤药物紫杉醇或抗疟疾青蒿素，或对人类和动物健康有害的，如广泛存在的霉菌毒素。许多萜类化合物是已知的植物或真菌激素、植物保护剂或微生物毒力因子。广泛存在的挥发性细菌有机物Geosmin和甲基异龙脑是湿润土壤特有气味的原因，也是公共饮用水供应和水产养殖中周期性、广泛发生和代价高昂的难闻味道和气味的原因。除了萜类代谢产物的内在生物学意义外，微生物萜类生物化学、结构生物学和分子遗传学的研究为发现新的生化反应和机制，理解天然产物生物合成途径的分子逻辑，阐明调控这些途径表达的未知因素，以及协调应用生化、结构生物学、生物信息学和分子遗传学工具了解微生物代谢提供了独特的平台。

DESCRIPTION (provided by applicant): The biosynthesis, mechanistic enzymology and molecular genetics of terpenoid biosynthesis in bacteria and fungi will be investigated, with particular emphasis on the mechanistic and structural characterization of three broad classes of proteins uncovered by mining of genomic databases and functional gene expression that present novel structural, mechanistic, or biological features: 1) Investigations of terpene synthases will focus on defining the cyclization mechanisms and elucidating the role of each protein in catalyzing the formation of its individual cyclization products. A combination of bioinformatic tools as well as mutational approaches will be used to identify targets that represent new structural types or unusual biosynthetic reactions and mechanisms, or that correspond to cryptic synthases of currently unknown function that are implicated in biologically important processes such as fungal virulence. The mechanistic focus will be on discovering new cyclases and on unraveling the protein structural basis for catalysis and product specificity using a combination of site-directed mutagenesis and protein crystallography. 2) A variety of catalytically novel oxygenases will be investigated that play key roles in the metabolic conversion of terpene hydrocarbons and alcohols to biosynthetic end-products. 3) The regulation of microbial terpene biosynthesis will also be probed, focusing on two intriguing model systems, the regulation of the biosynthesis of the antibiotic pentalenolactone and the exploration of the cryptic role of a highly conserved nucleotide-binding protein associated with the biosynthetic genes for the widely occurring volatile organic metabolite 2-methylisoborneol. Tens of thousands of terpenoid natural products are present in both terrestrial and marine plants, in fungi and liverworts, and numerous bacteria. Many of these compounds are widely used in human medicine such as the antitumor agent taxol or the antimalarial artemisinin, or harmful to human and animal health, such as the widely occurring trichothecene mycotoxins. Many terpenoids are known plant or fungal hormones, plant protectants, or microbial virulence factors. The ubiquitous volatile bacterial organics geosmin and methylisoborneol, which are responsible for the characteristic odor of moist soil, are also the cause of periodic, widely occurring, and costly episodes of unpleasant taste and odor in public drinking water supplies and in aquaculture. Beyond the intrinsic biological importance of terpenoid metabolites, the study of microbial terpene biochemistry, structural biology, and molecular genetics provides a unique platform for the discovery of new biochemical reactions and mechanisms, for the understanding of the molecular logic of natural product biosynthetic pathways, for the elucidation of the still obscure factors regulating expression of these pathways, and for the coordinated application of biochemical, structural biological, bioinformatic, and molecular genetics tools to the understanding of microbial metabolism.