Biosynthesis of Monoterpenes and Sesquiterpenes

单萜和倍半萜是植物产物的类异戊二烯家族的C10和C15成员。腺毛提取的选择性技术和亲和层析方法允许以高产率纯化萜烯代谢的酶。本研究测试了焦磷酸香叶酯异构化-环化偶联生成对称单萜的立体化学模型。立体特异性标记的前体，选择性的化学降解方案和对映异构体的色谱分离，允许神秘的反应步骤的立体化学，和cross-shift模式，被破译。四种类型的倍半萜环化酶被用来比较倍半萜生物合成的机制，立体化学和酶学。细胞色素P450依赖性羟化酶催化母体环萜类化合物氧化转化的第一步;并且已经分离出这些高度特异性的混合功能氧化酶中的几种。这些微粒体系统将被溶解并分离成组分，并将采用复溶实验、动力学分析和结合研究来建立观察到的极端底物选择性的基础。将设计有效的、高选择性的羟化酶抑制剂，其沿着环化的特异性抑制剂，将用于在体内控制萜烯的产生。许多生物分子和天然产物都是由基本的五碳结构单元组成的。萜烯是由这种结构单元制成的化合物，其赋予许多植物芳香，使其可用作香料、香料和昆虫引诱剂或驱虫剂。这项研究将提高我们对萜烯代谢的理解，有助于尝试利用涉及萜类化合物的生态相互作用，并有助于努力提高这些具有商业价值的天然产品的生产。

Monoterpenes and sesquiterpenes are the C10 and C15 members of the isoprenoid family of plant products. Selective techniques for extraction of glandular trichomes and affinity chromatography methods allow purification of the enzymes of terpene metabolism in high yield. This research tests a stereochemical model for the coupled isomerization- cyclization of geranyl pyrophosphate to symmetrical monoterpenes. Stereospecifically labeled precursors, selective chemical degradation schemes and chromatographic resolution of enantiomers, permit the cryptic stereochemistries of the reaction steps, and hydride-shift patterns, to be deciphered. Four types of sesquiterpene cyclases are being used to compare the mechanism, stereochemistry and enzymology of sesquiterpene biosynthesis. Cytochrome P450-dependent hydroxylases catalyze the first step in the oxidative transformation of parent cyclic terpenoids; and several of these highly specific mixed- function oxidases have been isolated. These microsomal systems will be solubilized and separated into components, and reconstitution experiments, kinetic analysis, and binding studies will be employed to establish the basis for the extreme substrate selectivity observed. Potent, highly selective inhibitors of the hydroxylases will be designed which, along with a specific inhibitor of cyclization, will be used to manipulate terpene production in vivo. %%% Many biological molecules and natural products are composed of a basic five-carbon building block. Terpenes are the compounds made from this building block that impart fragrances to many plants making them useful as perfume, spices and attractants or repellants to insects. This research will improve our understanding of terpene metabolism, aid in attempts to exploit ecological interactions involving terpenoids, and assist in efforts to improve production of these commercially valuable natural products.