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大量人类药物的已证明记录。在天然产物中，萜类化合物(异戊二烯)是最大的一类(已知为50,000种)，这里所针对的20碳二萜类化合物构成了这些化合物中相当大的一部分(&gt；12,000已知)。事实上，二萜类化合物的广泛多样性表明，这种C20主干可以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/紫杉醇TM和抗生物多替林)或研究工具(例如蛋白激酶C激活和促进肿瘤的佛波醇酯，以及腺苷环化酶激活剂forsklin)。相反，我们最近发现，人类病原体结核分枝杆菌利用一种二萜类化合物--异结核菌素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对来自植物和微生物的酶的生产性研究，这些酶催化形成潜在的碳氢化合物骨架结构。具体地说，我们将建立在上一次授权期的发现基础上，其中不仅包括异结核菌素的发现，而且还包括一些第一个二萜合成酶的晶体结构的阐明。这项提议推进了我们的长期目标，即设计酶和代谢途径，以生产靶向文库和特定的单一萜类“天然”产品，用于药物研究和使用。特别是，这项建议的目标是对催化形成二萜类天然产物结构多样性的碳氢骨架的合成酶/环合酶进行详细的结构和功能研究。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获得此类天然产品的机会有影响，而且对潜在的药物靶点，特别是在广泛存在的病原体结核杆菌中具有洞察力。

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.