Enzymatic Cyclization to Labdanes and Related Diterpenoid Natural Products

描述（由申请人提供）：Labdane和相关的二萜类化合物形成了一大组近7，000种有机化合物，显示出广泛的生物活性，包括许多具有显著药物活性，特别是医学相关的抗癌，抗炎或抗微生物作用。此外，许多这些普遍稀缺的天然产品的潜在用途尚未得到充分探索。这些复杂化合物的生物合成是通过一个知之甚少的酸/碱催化（II类）反应，形成双环核心结构，定义了这个超家族的天然产物。然后，通过更典型的烯丙基二磷酸离子化驱动（I类）反应具体阐述该双环核心。有趣的是，这些机制上不同的反应介导的遗传相关的萜烯脱氢酶，虽然在不同的活性位点。尽管两类劳丹相关的二萜类糖苷酶在启动大量天然产物的生物合成中具有重要的功能，但对底物和产物特异性的酶决定簇知之甚少。我们建议研究这些连续的环化反应，作为我们长期目标工程萜类天然产物生物合成用于制药目的的第一步。在我们以前发表的和初步的研究的基础上，我们假设，I类活性位点位于C-末端结构域，而II类活性位点位于两个其他结构域之间的界面。与I类反应相关的结构定义的C-末端结构域使得能够通过大分子建模指导的突变分析立即研究观察到的特异性。为了表征II类环化更基本的机械酶学研究提出了确定催化的决定因素，以及初步调查的产品特异性。此外，对于这两类反应，我们希望通过合作实验结构测定，并通过继续鉴定功能新颖的酶，沿着初步的代谢工程研究，获得显着的见解。因此，拟议的研究将阐明的结构-功能关系的基础上的连续I类/I类环化反应劳丹相关的二萜生物合成，并将有更广泛的影响，进一步提高我们的理解和使用萜类化合物酶促环化更普遍。

DESCRIPTION (provided by applicant): Labdanes and related diterpenoids form a large group of almost 7,000 organic compounds that display a wide range of biological activity and includes a number with significant pharmaceutical activity, particularly medically relevant anticancer, anti-inflammatory, or antimicrobial effects. In addition, potential uses for many of these generally scarce natural products have yet to be fully explored. Biosynthesis of these complex compounds is initiated via a poorly understood acid/base catalyzed (class II) reaction, which forms the bicyclic core structure that defines this super-family of natural products. This bicyclic core is then specifically elaborated by a more typical allylic diphosphate ionization driven (class I) reaction. Interestingly, these mechanistically distinct reactions are mediated by phylogenetically related terpene synthases, albeit in different active sites. Despite the functional importance of the two classes of labdane-related diterpene synthases in initiating the biosynthesis of large numbers of natural products with both realized and potential medical significance, little is known about the enzymatic determinants underlying substrate and product specificity. We propose to investigate these consecutive cyclization reactions as a first step towards our long-term goal of engineering terpenoid natural product biosynthesis for pharmaceutical purposes. On the basis of our previously published and preliminary studies, we hypothesize that the class I active site resides in the C-terminal domain, while the class II active site lies at the interface between two other domains. The structurally defined C-terminal domain associated with class I reactions enables immediate study of the observed specificity via macromolecular modeling directed mutational analysis. To characterize class II cyclization more basic mechanistic enzymology studies are proposed to identify the determinants for catalysis, as well as initial investigations of product specificity. In addition, for both classes of reactions we expect to obtain significant insights through collaborative experimental structure determination, and via continued identification of functionally novel enzymes, along with initial metabolic engineering studies. Thus, the proposed studies will elucidate the structure-function relationships underlying the consecutive class I I/class I cyclization reactions in labdane-related diterpene biosynthesis, and will have a broader impact in further increasing our understanding and use of terpenoid enzymatic cyclization more generally.