Enzymatic Cyclization to Labdanes and Related Diterpenoid Natural Products

Labdane和相关的二萜类化合物形成了一个由近7,000种有机化合物组成的大类，它们显示出一种 广泛的生物活性，包括一些具有显著药理活性的物质，特别是 与医学相关的抗癌、抗炎或抗菌作用。此外，许多产品的潜在用途 在这些普遍稀缺的天然产品中，尚未得到充分开发。这些复合体的生物合成 化合物是通过一个鲜为人知的酸/碱催化的(II类)反应启动的，该反应形成 双环核心结构，定义了这一超家族的天然产品。然后这个双环核心被特别地 阐述了一个更典型的烯丙基二磷酸电离驱动(I类)反应。有趣的是，这些 机制上不同的反应是由系统发育相关的萜烯合成酶介导的，尽管在 不同的活动站点。尽管这两类与拉布丹相关的二萜在功能上很重要 合成酶在启动大量已实现和潜在天然产物生物合成中的作用 医学意义，对底物和产物背后的酶决定因素知之甚少 专一性。我们建议研究这些连续的环化反应，作为迈向我们 为制药目的设计萜类天然产物生物合成的长期目标。论 根据我们之前发表的和初步的研究，我们假设I类活动站点位于 在C-末端结构域中，而II类活性部位位于两个其他结构域之间的交界处。这个 与I类反应相关的结构上定义的C-末端结构域使立即研究 通过大分子模拟定向突变分析观察特异性。描述II类的特征 环化反应建议进行更基本的机械酶学研究，以确定决定因素 催化，以及对产品专一性的初步调查。此外，对于这两类反应，我们 期望通过协作性实验结构确定以及通过 继续鉴定具有新功能的酶，以及最初的代谢工程研究。因此， 拟议的研究将阐明连续类别背后的结构-功能关系 与拉布丹相关的二萜生物合成中的II/I类环化反应，并将在 进一步加深了我们对萜类化合物酶促环化的更广泛的理解和使用。

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 II/class I cyclization reactions in labdane-related diterpene biosynthesis, andwill have a broader impact in further increasing our understanding and use of terpenoid enzymatic cyclization more generally.