BIOSYNTHESIS OF TAXOL

紫杉醇的生物合成

• 批准号: 2096475
• 负责人: RODNEY B CROTEAU
• 金额: $ 14.84万
• 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/2096475
• 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， 从西部紫杉、短叶红豆杉和其它红豆杉属植物中分离出来的一种药物 物种 这种药物的天然来源非常有限。 任何改善紫杉醇生物学生产的尝试都需要 了解这种天然产物的生物合成和 路径的调节。 一个生物遗传方案已被提出， 关于确定的紫杉烷代谢物的出现和关于与 简单的单萜类化合物的生物合成转化;然而， 目前，没有实验支持的信息的生物合成 紫杉醇。 本项目的目标是确定数量，类型 和C20转化中的酶促步骤的顺序 类异戊二烯分支点中间体，香叶基香叶基焦磷酸， 紫杉醇，并确定该途径的限速步骤。 具体目标包括：（1）定义环化步骤 香叶基香叶基焦磷酸和烯烃前体的身份 （2）建立紫杉醇的羟基化序列 紫杉醇的路线上的烯烃，并破译乙酰化的顺序， 羟基和可能的生物合成功能， 氧杂环丁烷环的酶促反应路线 （4）确定后期的发生顺序和机制 在紫杉醇核的C-2和C-13处的酰化;和（5）确认 通过体内研究和描绘的速率限制步骤的途径 道路。 描述紫杉醇复杂途径的目标将 通过使用来自T. 和放射性标记前体的使用。 放射性标记产品 从早期的酶促反应将被用来指导 分离和鉴定相应的中间体， 红豆杉提取物，从其标记的底物用于随后的酶促反应， 将准备转型。 使用14 CO2的体内研究 然后在完整的树苗中进行标记以确认该途径， 结合同位素稀释分析，确定速率 限制步骤。 速率限制催化剂，和其他酶的 途径相关的半合成的努力，以生产紫杉醇从更多 容易获得的紫杉烷代谢产物，将是更详细的重点 研究作为遗传和分子方法的基础， 目标化合物的体内或体外生产得到改善。