Stereochemical Studies of Isoprenoid Biosynthesis

我们建议继续和扩大正在进行的研究的机制酶学和分子遗传学的萜类化合物的生物合成。重点将是催化通用无环前体法呢基二磷酸（FPP，1）环化为并环戊二烯（2）、马兜铃烯（3）、大根香叶醇（4）和紫穗槐二烯（5）的倍半萜烯糖苷酶家族，以及这些倍半萜烯代谢转化为抗生素如抗生素并环戊二烯内酯（6）或土臭素（7），链霉菌属的特征性气味成分。必须解决三个广泛且密切相关的问题：1）环化酶如何将特定的折叠模式或构象强加于其无环底物FPP和衍生的中间体？2)环化酶如何管理带正电荷的中间体，包括催化底物的初始电离， 稳定阳离子中间体，通过正电荷的猝灭终止反应？3）环化酶活性位点的性质是什么？为了回答这些问题并尽可能广泛地探索萜类化合物的作用机制和结构空间，我们设计了一套相互补充的实验方法，包括：A。X射线测定 与大卫W教授合作，研究了野生型和突变型萜类化合物脱氢酶的晶体结构，包括无底物和结合底物及中间类似物。Christianson（宾夕法尼亚大学）; B.基于基因组序列信息的细菌和真菌来源的新萜类化合物脱氢酶的分离、表达和机理研究;萜烯脱氢酶的定点诱变，利用这种突变体产生异常产物的混合物的趋势，所述异常产物是碳阳离子环化级联的通常隐蔽的中间体的诊断; D.萜烯作用机理研究 酶，使用同位素标记，同位素敏感的分支实验，和预稳态，快速化学淬灭动力学来确定从FPP形成单个倍半萜烯的机制; E. E.环倍半萜和萜类代谢产物的杂环化生产。杆菌与Jay D教授合作Keasling（加州大学伯克利分校），我们将使用大肠杆菌的工程菌株来表达单个倍半萜烯脱氢酶以及整个萜类生物合成基因簇。这 将利用实验系统生产10-100 mg量的萜类代谢物用于结构表征，并确定萜类生物合成途径中的全套基因、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.