TERPENE CYCLASES--FUNCTIONAL DOMAINS AND STRUCTURES

环萜类化合物在整个自然界中发现，但尤其包括 从植物中提取的一类重要的化合物， 具有抗生素、抗真菌和抗肿瘤活性的药剂。 的 环萜的生物合成由关键分支点酶决定 称为萜烯环化酶，或更恰当地称为萜烯脱氢酶。 的 本建议的目的是尽可能全面地了解 控制环化的结构、功能和化学特征 高度同源植物催化法呢基二磷酸 倍半萜环化酶和一种二萜环化酶。 拟议的工作依赖于 我们最近成功地用一个域交换策略来映射和替换 倍半萜环化酶基因之间的功能域，和酶学 细菌表达的环化酶蛋白的表征。 我们现在 建议使用野生型和突变型的新晶体学数据， 酶在具有结构域交换和位点定向的迭代过程中 诱变以更详细地鉴定那些结构域和氨基酸， 酸残基是催化特定部分步骤所必需的， 环化酶反应。 拟议的实验计划将提供 我们的长期目标的基础，重点是一个理性的基础上， 重新设计萜烯环化酶，用于酶促合成 药学上重要的萜类化合物或其合成前体。 这 结构/功能分析也应该增加我们的基本 对萜烯酶学和生物合成的一般认识，以及 胆固醇、类固醇激素、胆汁等环萜类化合物的生物合成 酸、类胡萝卜素和类维生素A、D、E和K 特别是。

Cyclic terpenoids are found throughout nature but comprise an especially important class of compounds from plants which serve as pharmaceutical agents with antibiotic, antifungal and antitumor activities. The biosynthesis of cyclic terpenes is determined by key branch point enzymes referred to as terpene cyclases, or more properly, terpene synthases. The objective of this proposal is to understand as completely as possible the structural, functional, and chemical features governing the cyclizations of farnesyl diphosphage (FPP) catalyzed by highly homologous plant sesquiterpene cyclases and one diterpene cyclase. The proposed work relies on our recent success with a domain-swapping strategy to map and substitute functional domains between sesquiterpene cyclase genes, and enzymological characterization of the bacterial expressed cyclase proteins. We are now proposing to use new crystallographic data of the wildtype and mutant enzymes in an iterative process with domain swapping and site directed mutagenesis to identify, in much greater detail, those domains and amino acid residues essential for catalysis of particular partial steps within the cyclase reactions. The proposed experimental plan will provide the foundation for our long range goal that focuses on a rationally based redesign of terpene cyclases for the enzymatically directed synthesis of pharmaceutically important terpenoids or their synthetic precursors. This structure/function analysis should also increase our fundamental appreciation for terpene enzymology and biosynthesis in general, and the biosynthesis of cyclic terpenoids like cholesterol, steroid hormones, bile acids, carotenoids and retinoids, and lipid soluble vitamins, A,D,E, and K in particular.