ENZYMATIC CYCLIZATION TO TERPENOID NATURAL PRODUCTS

酶促环化生成萜类天然产物

• 批准号: 2838481
• 负责人: RODNEY B CROTEAU
• 金额: $ 18.61万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838481
• 关键词: X ray crystallography; active sites; cyclization; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate analog; genetic library; isomerase; molecular cloning; monoterpenes; nucleic acid sequence; plant extracts; plant physiology; protein purification; protein structure function; recombinant proteins; site directed mutagenesis

DESCRIPTION: (Principla Investigator's): Monoterpene synthases provide the focus for study of prenyl diphosphate cyclization, the reaction of principal importance in C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A stereochemical model for the coupled isomerization-cyclization of the universal isoprenoid precursor, geranyl diphosphate, was developed through studies on the origin of the seven major monoterpene skeletal types. Selected synthases ((+)- and (-)-limonene synthases, (+)- and (-)-pinene synthases, and (+)- and (-)-bornyl diphosphate synthases from the same species (common sage) and from a phylogenetically distant source (grand fir)) that differ significantly in mechanistic and stereochemical features will be employed to examine active site structure-function relationships that underlie the formation of olefin isomers, oxygenated derivatives, and their enantiomers. Molecular cloning of (-)-4S-limonene synthase, catalyzing the simplest of all terpenoid cyclizations, has provided access to cDNAs encoding the other target synthases, and functional expression of the truncated enzymes, in which the troublesome plastidial transit peptides have been deleted, allows the examination of active site structure and function. The first two specific aims of the proposal are to utilize the similarity-based cloning strategy to acquire the remaining target cDNAs, and to express the appropriate truncations for high yield production of fully active 'pseudo-mature' enzymes for X-ray crystallographic studies and related investigations. In the third aim, the active sites of the recombinant synthases (with limonene synthase as the prototype) will be located using cysteine- and histidine-directed reagents and substrate protection/deprotection strategies, a mechanism-based alkylator, and photolabile substrate analogs to target hydrophobic binding pockets. Information from active site location, plus that gained by primary sequence comparisons, will be used in specific aim four to target selected residues and hydrophobic domains for mutagenesis. The mutants will be evaluated for kinetic behavior and product outcome to deduce which steps of the reaction cascade have been altered. In the final aim, substrate analogs will be used to examine the cryptic isomerization step of the reaction and to explore the catalytic repertoire of the cloned synthases. These studies will provide new information on the relationship of structure to reaction mechanism for these novel catalysts, and allow a clearer understanding of this important aspect of prenyl diphosphate metabolism.

描述：（主要调查员）：单萜合酶提供 重点用于研究前二磷酸酯环化，主体的反应 在众多萜类化合物的生物合成中，C-C键形成的重要性 具有药理意义的天然产物。 立体化学模型 用于通用类异戊二烯的耦合异构化周期化 前体，黄烷基二磷酸，是通过对起源的研究而开发的 在七种主要的单苯甲骨骼类型中。 选定的合成酶（（+） - 和 （ - ） - 柠檬烯合酶，（+） - 和（ - ） - Pinene合酶，（+） - 和 - （ - ） - 来自同一物种（共同鼠尾草）和 从系统发育遥远的来源（大冷杉）不同 在机械和立体化学特征上，将采用显着的 检查主动站点结构功能关系的关系，这是 烯烃异构体，氧化衍生物及其对映异构体的形成。 （ - ） - 4S-二烯烯合酶的分子克隆，催化最简单的 所有萜烯循环都提供了对编码另一个编码的cDNA的访问权限 靶标合酶和截短酶的功能表达，在 删除了麻烦的质质肽，允许 主动站点结构和功能的检查。 前两个 该提案的具体目的是利用基于相似性的克隆 获取其余目标cDNA的策略，并表达 适当的截断，用于高收率产生完全活跃的 用于X射线晶体学研究和相关的“伪成熟”酶 调查。 在第三个目标中，重组的活性位点 合成酶（将柠檬烯合酶作为原型）将使用 半胱氨酸和组氨酸指导的试剂和底物 保护/脱保护策略，一种基于机制的烷基机，以及 光值底物类似于靶向疏水结合口袋。 来自Active站点位置的信息，加上主要序列获得的信息 比较将用于特定目标四来靶向选定的残基 和疏水结构域用于诱变。 将评估突变体 动力学行为和产品结果来推断反应的哪些步骤 级联已经改变了。 在最终目标中，将使用底物类似物 检查反应的神秘异构化步骤并探索 克隆合酶的催化曲目。 这些研究将提供 有关结构与反应机制关系的新信息 这些新颖的催化剂，并可以更清楚地了解这一重要的 核二磷酸代谢的方面。