ENZYMATIC CYCLIZATION OF TERPENOID NATURAL PRODUCTS

萜类天然产物的酶促环化

• 批准号: 2176105
• 负责人: RODNEY B CROTEAU
• 金额: $ 16.61万
• 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 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176105
• 关键词: 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; site directed mutagenesis

Monoterpene cyclases (synthases) provide the focus for study of allylic pyrophosphate cyclization, a reaction type of major importance in C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A general stereochemical model has been proposed for the coupled isomerization-cyclization of the universal isoprenoid precursor, geranyl pyrophosphate, and key mechanistic elements of the scheme were confirmed through studies on the origin of the seven major monoterpene skeletal types. A selected set of cyclases [(+)- and (- )limonene synthase, (+)- and (-)-pinene synthase, and (+)- and (-)-bornyl pyrophosphate synthase] that differ significantly in mechanistic detail will be employed to examine active site structure-function relationships that underlie reaction variants. As the prototype, (-)-4S-limonene synthase was purified from isolated oil glands of Mentha and, from amino acid sequence information, degenerate oligonucleotide probes were prepared for screening an oil gland cDNA library, from which three full-length clones were isolated. These cDNA isolates were sequenced and verified by functional expression in Escherichia coil. Based on positive RNA blot hybridization and direct sequence comparison at the protein level, the (- )-limonene cyclase cDNA provides a powerful heterologous probe for isolating the cDNAs encoding the other monoterpene cyclases from the corresponding gland libraries of Salvia, Tanacetum and Citrus species. Where heterologous cDNA probing is not possible, an alternate strategy for purifying the target cyclase, and obtaining the gene, has been devised based on specific labeling of the protein with a mechanism-based inhibitor. A bacterial overexpression system based on the PET vector will be devised that permits rapid purification of the recombinant cyclases. The roles of active site cysteine and histidine residues in binding and catalysis were suggested by inhibition studies and by determining the protective influence of substrate and analogues representing different substrate binding domains. A photolabile substrate analogue was utilized for photoaffinity labeling of the presumptive hydrophobic pocket of the cyclases, and the mechanism-based inhibitor was used for labeling putative active site bases involved in terminating deprotonations. With these probes, labeled active site peptides will be generated for sequencing and location on the deduced primary structures. This information, plus that gained by primary sequence comparisons, will be used to target active site residues for mutagenesis. The mutant cyclases will be characterized with respect to kinetic behavior and product mixture, and a variety of biochemical techniques employed to deduce which step(s) of the complex reaction cascade have been altered. A series of substrate analogues will be used to examine the cryptic isomerization step of the reaction and to explore the catalytic repertoire of the cloned cyclases. The studies outlined should provide new information on the nature of these novel catalysts, particularly the relationship of enzyme structure to reaction mechanism, and allow a clearer understanding of this important aspect of prenyl pyrophosphate metabolism.

单苯乙烯环化酶（合酶）为研究烯丙基提供了重点 焦磷酸环化，这是C-C键中主要重要性的一种反应类型 在许多萜类天然产物的生物合成中形成 药理意义。一般立体化学模型已经 提出了通用的耦合异构化周期化的耦合异构化周期化 异普尼前体，焦磷酸香豆基和关键机械元素 该方案通过有关七个起源的研究得到了证实 主要的单苯乙烯骨骼类型。一组选定的循环酶[（+） - 和（ - ）柠檬烯合酶，（+） - 和（ - ） - Pinene合酶，（+） - 和（ - ） - 焦磷酸合酶]在机械细节上显着差异 将被用来检查主动站点结构功能关系 这是反应变体的基础。 作为原型，（ - ） - 4S-二烯 合成酶从Mentha的孤立油腺和氨基中纯化 酸序列信息，退化寡核苷酸探针 用于筛选油腺cDNA库，从中三个全长 克隆分离。测序这些cDNA分离株并通过 大肠盘线圈中的功能表达。 基于正RNA印迹 在蛋白质水平上的杂交和直接序列比较（ - ） - 丙烯环化酶cDNA提供了强大的异源探针 隔离从编码其他单单苯乙烯循环酶的cDNA 相应的萨尔维亚，塔纳西特和柑橘类的腺文库。 如果不可能进行异源cDNA探测，则是一种替代策略 已经设计了纯化靶环酶并获得基因 基于基于机制的蛋白质的特定标记 抑制剂。 基于PET载体的细菌过表达系统将 设计，允许快速纯化重组循环酶。 活性位点半胱氨酸和组氨酸残基在结合和 通过抑制研究和确定 底物和代表不同的类似物的保护作用 底物结合域。 使用了光值底物类似物 用于将其推定疏水口袋的光性标记 循环酶和基于机制的抑制剂用于标记推定 活跃的现场基础涉及终止去质子的依据。 与这些 将生成探针，标记为活性位点肽进行测序和 推导的主要结构上的位置。此信息，加上 通过一级序列比较获得，将用于靶向活跃位点 诱变的残基。突变循环酶的表征 尊重动力学行为和产品混合物，以及多种 用于推断复合体的哪个步骤所采用的生化技术 反应级联反应已改变。 一系列基板类似物将 用于检查反应的隐秘异构化步骤 探索克隆循环酶的催化曲目。 研究 概述应提供有关这些小说本质的新信息 催化剂，特别是酶结构与反应的关系 机制，并更清楚地了解 促头焦磷酸代谢。