Biosynthesis of Microbial Isoprenoids

描述（由申请人提供）：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和功能基因表达发现的三大类蛋白质的机制和结构表征，这些蛋白质呈现新的结构、机制或生物学特征：1）萜烯脱氢酶的研究将集中在定义环化机制和阐明每种蛋白质在催化其单独的环化产物形成中的作用。生物信息学工具以及突变方法的组合将用于鉴定代表新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的内切酶的靶标，所述隐蔽的内切酶涉及生物学上重要的过程，例如真菌毒力。机制的重点将是发现新的环化酶和解开催化和产品特异性的蛋白质结构基础，使用定点诱变和蛋白质晶体学的组合。2)将研究在萜烯烃和醇到生物合成终产物的代谢转化中起关键作用的各种催化新加氧酶。3)也将探讨微生物萜烯生物合成的调节，集中在两个有趣的模型系统，抗生素pentalenolactone的生物合成的调节和探索的神秘作用的高度保守的核苷酸结合蛋白与生物合成基因的广泛存在的挥发性有机代谢产物2-甲基异戊烯。 数以万计的萜类天然产物存在于陆生和海洋植物、真菌和苔类以及许多细菌中。这些化合物中的许多广泛用于人类医学，如抗肿瘤剂紫杉醇或抗疟疾剂青蒿素，或对人类和动物健康有害，如广泛存在的多孢霉烯真菌毒素。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。无处不在的挥发性细菌有机物土臭素和甲基异戊烯，这是负责潮湿土壤的特征气味，也是周期性的，广泛发生的，并在公共饮用水供应和水产养殖中的令人不快的味道和气味的昂贵事件的原因。除了萜类代谢物的内在生物学重要性之外，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物化学反应和机制、理解天然产物生物合成途径的分子逻辑、阐明调节这些途径的表达的仍然模糊的因子以及生物化学、生物化学和分子遗传学的协调应用提供了独特的平台。结构生物学，生物信息学和分子遗传学工具，以了解微生物代谢。

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.