STEROCHEMICAL STUDIES 0F ISOPRENOID BIOSYNTHESIS

倍半萜合成酶催化通用无环化合物的环化反应 前体法尼基二磷酸(1)到200个不同环中的任何一个 倍半萜碳氢化合物和醇，它们又可以作为 已知的数千种倍半萜类化合物的代谢前体。 这些代谢物可以表现出广泛的抗生素或其他 生理活性，无论是抗病毒，抗菌，抗真菌， 杀虫、拒食、细胞抑制、免疫抑制或神经毒性。 对两种立体特异性标记底物进行了广泛的研究 以及各种竞争性抑制剂，在我们自己的和 其他实验室，已经导致了一种通用的机械化和立体化学 萜类环化模型。 我们建议继续和扩大正在进行的对机械装置的研究 萜类环化反应的酶学和分子遗传学。焦点 将在微生物倍半萜合成酶家族上催化 法尼基二磷酸(1)转化为三氯二烯(2)， 马兜铃烯(3)、β-反式佛手萝卜素(4)和表铜酚(5)。在……里面 与华盛顿州立大学罗德尼·克罗托教授合作 我们还将研究一组单萜合成酶，包括柠檬烯。 (6)合酶。为了测试并进一步扩展通用的 三个广泛而紧密的萜类环化反应的立体化学模型 必须解决相互关联的问题：1)循环系统如何施加 底物FPP上的特定折叠图案或构象并衍生 中间体？2)环化酶如何管理带电的中间体， 包括对底物初始电离的催化，通过 阳离子中间体的稳定，直到反应的终止 正电荷猝灭？一个相关的问题是环化酶是如何 避免因与高度的 它必须容纳亲电物种？3)它的性质是什么 活动站点？ 为了回答这些问题，我们设计了一套相辅相成的 实验方法，涉及：A.合成和环化 立体标记底物和中间体及其分析 ~2H和~2H标记在环化产物中的分布 13C核磁共振谱；序列萜类环化酶的纯化及克隆， 测序及相关结构基因的过表达； 底物和中间体类似物的设计、合成和测试 正常环化的竞争性抑制物或活性部位 倍半萜的定向和基于机理的不可逆失活剂 单萜烯合酶；D.正常酶的分离和鉴定 通过快速淬火实验或使用 只能进行部分反应的反常底物。 建议中开发的技术和实验方法 研究有望广泛适用于对NOT的理解 只有萜类环化，但酶机制和生物合成 一般调查，以及天然产品的演变 生物合成途径。

Sesquiterpene synthases catalyze the cyclization of the universal acyclic precursor, farnesyl diphosphate (1) to any of 200 distinct cyclic sesquiterpene hydrocarbons and alcohols, which in turn can serve as the metabolic precursors of the many thousands of known sesquiterpenoids. These metabolites can display a wide range of antibiotic or other physiological activities, be it antiviral, antibacterial, antifungal, insecticidal, antifeedant, cytostatic, immunosuppressive, or neurotoxic. Extensive investigations with both stereospecifically labeled substrates as well as with various competitive inhibitors, carried out in our own and other laboratories, have led to a general mechanistic and stereochemical model of terpenoid cyclization. We propose to continue and extend ongoing studies of the mechanistic enzymology and molecular genetics of terpenoid cyclizations. The focus will be on a family of microbial sesquiterpene synthases which catalyze the conversion of farnesyl diphosphate (1) to trichodiene (2), aristolochene (3) beta-trans-bergamotene (4), and epi-cubenol (5). In collaboration with Professor Rodney Croteau of Washington State University we will also study a group of monoterpene synthases, including limonene (6) synthase. In order to test and to extend further a general stereochemical model of terpenoid cyclizations, three broad and closely interrelated questions must be addressed: 1) How does a cyclase impose a specific folding pattern or conformation on the substrate FPP and derived intermediates? 2) How does a cyclase manage charged intermediates, including.catalysis of the initial ionization of substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? A related issue is how does the cyclase avoid annihilation which would result from reaction with the highly electrophilic species it must accommodate? 3) What is the nature of the active site? To answer these questions, we have devised a set of mutually complementary experimental approaches, involving: A. Synthesis and cyclization of stereospecifically labeled substrates and intermediates and analysis of the distribution of label in the derived cyclization products by 2H and 13C NMR; B . Purification of sequiterpene cyclases and cloning, sequencing, and overexpression of the relevant structural genes; C. Design, synthesis, and testing of substrate and intermediate analogs as either competitive inhibitors of the normal cyclization or active-site directed and mechanism-based irreversible inactivators of sesquiterpene or monoterpene synthases; D. Isolation and identification of normally enzyme- bound intermediates through rapid quench experiments or the use of anomalous substrates which can only undergo partial reactions. The techniques and experimental approaches developed in the proposed research are expected to be broadly applicable to the understanding of not only terpenoid cyclizations but enzyme mechanistic and biosynthetic investigations in general, as well as the evolution of natural products biosynthetic pathways.