BIOSYNTHESIS OF TAXOL

紫杉醇的生物合成

• 批准号: 3199750
• 负责人: RODNEY B CROTEAU
• 金额: $ 13.72万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3199750
• 关键词: Conifera; acetylation; acylation; antineoplastics; biosynthesis; cell free system; chemical kinetics; chemical structure; cyclization; diterpenes; enzyme mechanism; esterification; gas chromatography; high performance liquid chromatography; hydroxyl group; infrared spectrometry; mass spectrometry; medicinal plants; nuclear magnetic resonance spectroscopy; paclitaxel; plant extracts; plant proteins; radiotracer; ultraviolet spectrometry

Taxol, a highly functionalized diterpenoid, is an important antitumor drug isolated from the Western Yew, Taxus brevifolia, and other Taxus species. The supply of this drug from natural sources is very limited. Any attempt to improve the biological production of taxol requires an understanding of the biosynthesis of this natural product and of the regulation of the pathway. A biogenetic scheme has been proposed based on the occurrence of defined taxane metabolites and on analogy to biosynthetic transformations of simpler monoterpenoids; however, there is at present no experimentally supported information on the biosynthesis of taxol. The objective of this project is to determine the number, types and sequence of enzymatic steps in the transformation of the C20 isoprenoid branch-point intermediate, geranylgeranyl pyrophosphate, to taxol, and to determine the rate limiting step(s) of the pathway. Specific aims include: (1) defining the cyclization step of geranylgeranyl pyrophosphate and the identity of the olefin precursor of taxol; (2) establishing the sequence of hydroxylations of the parent olefin on route to taxol, and deciphering the sequence of acetylations of the hydroxyl groups and the possible biosynthetic function of the acetylation process; (3) elucidating the enzymatic route of oxetane ring formation; (4) determining the order and mechanism of late stage acylations at C-2 and C-13 of the taxol nucleus; and (5) confirming the pathway by in vivo studies and delineating the rate limiting step(s) of the pathway. This goal of describing the complex pathway to taxol will be accomplished through the use of cell-free enzyme systems from T. brevifolia and the use of radiolabeled precursors. Radiolabeled products from the early enzymatic reactions will be employed to direct the isolation and identification of the corresponding intermediates from Taxus extracts, from which labeled substrates for succeeding enzymatic transformations will be prepared. In vivo studies employing 14CO2 labeling in intact saplings will then be used to confirm the pathway and, in combination with isotopic dilution analysis, to determine the rate limiting step(s). The rate limiting catalyst, and other enzymes of the pathway relevant to semi-synthetic efforts to produce taxol from more readily available taxane metabolites, will be the focus of more detailed study as the foundation for genetic and molecular approaches to the improved in vivo or in vitro production of the target compound.

紫杉醇是一种高度功能化的二萜类化合物，是一种重要的抗肿瘤药物 从西部红豆杉、短叶红豆杉和其他红豆杉中分离出的药物 物种。这种药物的天然来源供应非常有限。 任何提高紫杉醇生物产量的尝试都需要 对这一天然产物的生物合成和对 对该途径的调控。一种生物遗传方案已经被提出基于 关于确定的紫杉烷代谢物的存在以及类比 简单的单萜类化合物的生物合成转化；然而，有 目前还没有关于生物合成的实验支持的信息 紫杉醇。这个项目的目标是确定数量、类型 和C20转化中的酶步骤序列 异戊二烯支点中间体香叶基焦磷酸 紫杉醇，并确定该途径的限速步骤(S)。 具体目标包括：(1)确定环化步骤 香叶基香叶基焦磷酸及其烯烃前体的鉴定 紫杉醇；(2)建立母体的羟基化序列 合成紫杉醇的路线上的烯烃，以及破译紫杉醇的乙酰化序列 羟基及其可能的生物合成功能 乙酰化反应；(3)氧杂环酶促反应路线的确定 形成；(4)确定后期的顺序和机制 紫杉醇核的C-2和C-13上的酰化反应；和(5)确认 通过体内研究和描绘的速度限制步骤(S)的途径 这条小路。描述紫杉醇的复杂途径的这一目标将 通过使用T. 短叶植物和放射性标记前体的使用。有放射性标记的产品 从早期的酶反应将被用来指导 中草药中相应中间体的分离与鉴定 红豆杉提取物，用于后续酶促反应的标记底物 将准备好转型。使用14CO2的体内研究 然后，将使用完整树苗的标记来确认路径， 结合同位素稀释分析，确定 限步(S)。限速催化剂和其他酶的作用 与半合成努力相关的途径，以从更多的 随手可得的紫杉烷代谢物，将对重点进行更详细的阐述 作为遗传学和分子生物学方法的基础的研究 改进了目标化合物的体内或体外生产。