Enzymatic formation of the hydrocarbon skeletons underlying diterpenoid diversity

描述（由申请人提供）：天然产品具有直接或作为先导化合物提供人用药物的重要部分的证明记录。在天然产物中，萜类（类异戊二烯）作为最大的类别（已知> 50，000种）而突出，其中20-碳二萜类在此形成这些（已知> 12，000种）中的显著部分。事实上，二萜类化合物的广泛多样化表明，可以由这种C20主链形成的多种结构提供了丰富的生物活性来源，并且许多这些天然产物被用作药物（例如，抗癌紫杉醇/TaxolTM和抗生素mutilin）或研究工具（例如，蛋白激酶C激活和促肿瘤的佛波醇酯，和腺苷酸环化酶激活剂毛喉素）。相反，我们最近发现人类病原体结核分枝杆菌利用二萜类化合物isotuberculosinol来调节我们对感染的初始免疫反应。因此，我们建议在这里继续我们的生产性研究的酶，从植物和微生物，催化形成的基本碳氢化合物骨架结构。具体来说，我们将建立在我们的发现从以前的补助金期间，其中不仅包括isotuberculosinol的发现，但阐明了一些第一个二萜合酶晶体结构以及。然后，该提议推进了我们的长期目标，即工程化酶和代谢途径，用于生产靶向文库和特定的单个萜类化合物“天然”产物，用于药物研究和使用。特别是，这个建议的目的是一个详细的结构-功能的调查，催化形成的碳氢化合物骨架的二萜类天然产物的结构多样性的脱氢酶/环化酶。这些酶催化复杂的亲电反应，形成新的碳-碳键，并在启动二萜类化合物的生物合成中发挥重要作用。因此，我们的研究不仅对提供更多的天然产品具有意义，而且对了解潜在的药物靶点，特别是在广泛分布的病原体M中具有意义。结核

DESCRIPTION (provided by applicant): Natural products have a proven record of providing a significant fraction, either directly or as lead compounds, of human medicines. Among natural products, the terpenoids (isoprenoids) stand out as being the largest class (>50,000 already known), with the 20-carbon diterpenoids targeted here forming a significant fraction of these (>12,000 known). Indeed, the extensive diversification of diterpenoids indicates that the manifold structures that can be formed from this C20 backbone provide a rich source of biological activity, and a number of these natural products are used as pharmaceuticals (e.g., the anti- cancer paclitaxel/TaxolTM and anti-biotic mutilins) or research tools (e.g., the protein kinase C activating and tumor promoting phorbol esters, and adenylate cyclase activator forskolin). Conversely, we have recently discovered that the human pathogen Mycobacterium tuberculosis utilizes a diterpenoid, isotuberculosinol, to modulate our initial immune response to infection. Accordingly, we propose here to continue our productive investigations of the enzymes, from both plants and microbes, that catalyze formation of the underlying hydrocarbon skeletal structures. Specifically, we will build on our findings from the previous grant period, which includes not only the discovery of isotuberculosinol, but elucidation of some of the first diterpene synthase crystal structures as well. This proposal then advances our long- term goal of engineering enzymes and metabolic pathways for the production of targeted libraries and specific individual terpenoid 'natural' products for pharmaceutical investigation and use. In particular, the objective of this proposal is a detailed structure-function investigation of the synthases/cyclases that catalyze formation of the hydrocarbon skeletons underlying the structural diversity of diterpenoid natural products. These enzymes catalyze complex electrophilic reactions that form new carbon-carbon bonds, and play important roles in initiating diterpenoid biosynthesis. Accordingly, our studies have implications not only for providing increased access to such natural products, but also insights into potential drug targets, specifically in the widespread pathogen M. tuberculosis.