TERPENE CYCLASES--FUNCTIONAL DOMAINS AND STRUCTURES

环萜类化合物在自然界中广泛存在，但含有一种特殊的 从用作药物的植物中提取的一类重要化合物 具有抗菌、抗真菌和抗肿瘤活性的药物。这个 环萜烯的生物合成由关键分支点酶决定 被称为萜烯环酶，或者更准确地说，萜烯合成酶。这个 这项建议的目标是尽可能全面地了解 环化反应的结构、功能和化学特征 高度同源植物催化的法尼基二磷酸盐(FPP) 倍半萜环化酶和一个二萜环化酶。拟议的工作依赖于 关于我们最近成功地使用域交换策略来映射和替换 倍半萜环化酶基因之间的功能结构域和酶学 细菌表达的环化酶蛋白的鉴定。我们现在是 建议使用野生型和突变型的新的结晶学数据 结构域交换和定点定向的迭代过程中的酶 突变以更详细地识别这些结构域和氨基酸 酸性残基是催化特定部分步骤所必需的 环酶反应。拟议的试验计划将提供 为我们的长期目标奠定基础，专注于基于理性的 用于酶促合成的萜烯环化酶的重新设计 具有重要药用价值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础 对萜烯的酶学和生物合成的一般评价，以及 胆固醇、类固醇激素、胆汁等环状萜类化合物的生物合成 酸、类胡萝卜素和维甲酸，以及脂溶维生素A、D、E和K 尤其是。

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.