STEROCHEMICAL STUDIES 0F ISOPRENOID BIOSYNTHESIS

倍半萜烯糖苷酶催化通用的非环状结构的环化， 前体，法呢基二磷酸（1）到任何200个不同的环状 倍半萜烯烃和醇，其又可以用作 它是数千种已知倍半萜类化合物的代谢前体。 这些代谢物可以显示出广泛的抗生素或其他生物活性。 生理活性，无论是抗病毒，抗菌，抗真菌， 杀虫、拒食、细胞生长抑制、免疫抑制或神经毒性。 用两种立体特异性标记底物进行广泛研究 以及各种竞争性抑制剂，在我们自己的， 其他实验室，已经导致了一般的机械和立体化学 萜类化合物环化模型。 我们建议继续和扩大正在进行的机制研究， 萜类化合物环化的酶学和分子遗传学。重点 将是一个微生物倍半萜烯脱氢酶家族， 将法呢基二磷酸（1）转化为α-二烯（2）， 马兜铃烯（3）、β-反式-香柠檬烯（4）和表-古巴烯醇（5）。在 与华盛顿州立大学的Rodney Croteau教授合作 我们还将研究一组单萜脱氢酶，包括柠檬烯 (6)合成酶为了测试和进一步扩展一个通用的 萜类化合物环化立体化学模型，三个广泛而紧密的 必须解决相互关联的问题：1）环化酶如何施加 在基底FPP上的特定折叠图案或构象， 中间体？2)环化酶如何管理带电的中间体， 包括催化底物的初始电离，通过 阳离子中间体的稳定化，直至反应终止， 正电荷的猝灭一个相关的问题是环化酶如何 避免由于与高分子反应而导致的湮灭。 必须容纳的亲电物种3)的本质是什么 活动场所？ 为了回答这些问题，我们设计了一套相互补充的 实验方法，涉及：A.的合成及环化反应 立体特异性标记的底物和中间体及其分析 标记在衍生的环化产物中通过2 H的分布， 13 C NMR; B .半萜环化酶的纯化和克隆， 测序和相关结构基因的过表达; C. 设计、合成和测试底物和中间体类似物， 正常环化或活性位点的竞争性抑制剂 定向的和基于机理的不可逆的倍半萜烯或 单萜木聚糖酶; D.正常酶的分离和鉴定 通过快速淬灭实验或使用 只能发生部分反应的异常基质。 提出的技术和实验方法， 研究预计将广泛适用于了解不 只有萜类化合物环化，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.