Stereochemical Studies of Isoprenoid Biosynthesis

我们建议继续和扩大正在进行的关于萜类化合物生物合成的机械酶和分子遗传学的研究。重点将集中在倍半萜合成酶家族上，该家族催化通用的无环前体法尼基二磷酸(FPP，1)环化为五茂烯(2)、马兜铃烯(3)、杰马卡二烯(4)和氨甲二烯(5)，以及这些倍半萜的代谢转化为抗生素，如抗生素戊烯内酯(6)或地黄素(7)，链霉菌物种的特征含氧组分。必须解决三个广泛且密切相关的问题：1)环酶如何在其非环底物fpp及其衍生中间体上施加特定的折叠模式或构象？2)环酶如何管理带正电的中间体，包括催化底物的初始电离。 阳离子中间体的稳定，直到正电荷猝灭反应的终止？3)环化酶活性部位的性质是什么？为了回答这些问题，并尽可能广泛地探索萜类化合物的机理和结构空间，我们设计了一套相互补充的实验方法，包括：a.X射线的测定 与David W.Christian教授(宾夕法尼亚大学)合作，研究野生型和突变型萜类合成酶的晶体结构，包括无底物和结合底物和中间类似物；B.基于基因组序列信息从细菌和真菌来源分离、表达和研究新的萜类合成酶；c.对萜类合成酶进行定点突变，利用这种突变产生异常产物混合物的趋势，以诊断碳阳离子环化级联反应的通常隐蔽的中间产物；D.萜烯的机理研究 合成酶，使用同位素标记，同位素敏感的分支实验，和稳定前的快速化学猝灭动力学来确定从FPP形成单个倍半萜的机制；E.在大肠杆菌中异源生产环倍半萜和萜类代谢物。与加州大学伯克利分校的Jay D.Keasling教授合作，我们将使用工程菌株大肠杆菌表达单个倍半萜合成酶以及整个萜类生物合成基因簇。这 将开发实验系统来生产10-100毫克量的萜类代谢物，用于结构表征和确定萜类生物合成途径中的一整套基因、酶和代谢中间体。

We propose to continue and extend ongoing studies of the mechanistic enzymology and molecular genetics of terpenoid biosynthesis. The focus will be on a family of sesquiterpene synthases which catalyze the cyclization of the universal acyclic precursor farnesyl diphosphate (FPP, 1) to pentalenene (2), aristolochene (3), germacradienol (4), and amorphadiene (5), as well as the metabolic conversion of these sesquiterpenes to antibiotics such as the antibiotic pentalenolactone (6) or to geosmin (7), the characteristic oderiferous constituent of Streptomyces species. Three broad and closely interrelated questions must be addressed: 1) How does a cyclase impose a specific folding pattern or conformation on its acyclic substrate FPP and derived intermediates? 2) How does a cyclase manage positively charged intermediates, including catalysis of the initial ionization of the substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? and 3) What is the nature of the cyclase active site? To answer these questions and to explore terpenoid mechanism and structure space as broadly as possible, we have devised a set of mutually complementary experimental approaches involving : A. Determination of the X-ray crystallographic structures of wild-type and mutant terpenoid synthases, both substrate-free and with bound substrate and intermediate analogs, in collaboration with Prof. David W. Christianson (University of Pennsylvania); B. Isolation, expression and mechanistic investigation of new terpenoid synthases from bacterial and fungal sources based on genomic sequence information; C. Site-directed mutagenesis of terpene synthases, exploiting the tendency of such mutants to produce mixtures of aberrant products that are diagnostic of the normally cryptic intermediates of the carbocationic cyclization cascade; D. Mechanistic studies of terpene synthases, using isotopic labeling, isotopically sensitive branching experiments, and pre-steady state, rapid chemical quench kinetics to define the mechanism of formation of individual sesquiterpenes from FPP; E. Heterologous production of cyclic sesquiterpenes and terpenoid metabolites in E. coli. In collaboration with Prof. Jay D. Keasling (University of California, Berkeley), we will use an engineered strain of Escherichia coli to express individual sesquiterpene synthases as well as entire terpenoid biosynthetic gene clusters. This experimental system will be exploited to produce 10-100 mg quantities of terpenoid metabolites for structural characterization and to define the complete set of genes, enzymes, and metabolic intermediates in terpenoid biosynthetic pathways.