Enzymatic Cyclization to Labdanes and Related Diterpenoid Natural Products

描述(申请人提供)：Labdanes和相关的二萜类化合物形成了一大类近7,000种有机化合物，显示出广泛的生物活性，并包括一些具有显著药理活性的化合物，特别是在医学上相关的抗癌、抗炎或抗菌作用。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开发。这些复杂化合物的生物合成是通过一个鲜为人知的酸/碱催化(II类)反应启动的，该反应形成了定义这一超级天然产物家族的双环核心结构。然后，通过更典型的烯丙基二磷酸电离驱动(I类)反应来具体阐述该双环核。有趣的是，这些机械上不同的反应是由系统发育相关的萜烯合成酶介导的，尽管在不同的活性部位。尽管这两类与拉布丹相关的二萜合成酶在启动大量具有已实现和潜在医学意义的天然产物的生物合成中具有重要的功能，但人们对底物和产物专一性的酶决定因素知之甚少。我们建议研究这些连续的环化反应，作为实现我们的长期目标的第一步，即设计用于制药目的的萜类天然产物的生物合成。根据我们以前发表的和初步的研究，我们假设I类活性中心位于C-末端结构域，而II类活性中心位于其他两个结构域的交界处。与I类反应相关的结构定义的C-末端结构域使通过大分子建模指导突变分析立即研究观察到的特异性成为可能。为了确定II类环化反应的特征，建议进行更基础的机械性酶学研究，以确定催化的决定因素，以及对产物专一性的初步调查。此外，对于这两类反应，我们希望通过合作的实验结构确定，以及通过继续鉴定功能新颖的酶，以及初步的代谢工程研究，获得重要的见解。因此，这些研究将阐明与拉布丹相关的二萜生物合成中连续的I类I/I类环化反应背后的结构-功能关系，并将对进一步加深我们对萜类化合物酶促环化的更广泛的理解和使用具有更广泛的影响。

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.