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.

紫杉醇是一种高度官能化的二萜，是重要的抗肿瘤 与西紫杉，出租车Brevifolia和其他出租车隔离的药物 物种。 从天然来源提供这种药物的供应非常有限。 任何改善紫杉醇生物生产的尝试都需要 理解这种天然产品的生物合成和 路径的调节。 已经提出了基于生物遗传学的方案 关于定义的紫杉烷代谢产物的发生，类比 较简单的单苯甲酸酯的生物合成转化；但是，有 目前尚无有关生物合成的实验支持信息 紫杉醇。 该项目的目的是确定数量，类型 和C20转换中的酶促步骤的顺序 类异型分支点中间体，天形凝集丙基磷酸盐，至 紫杉醇，并确定途径的限制步骤。 具体目的包括：（1）定义的环化步骤 香烷基焦磷酸和烯烃前体的身份 紫杉醇； （2）建立父母的羟基序列 烯烃在通往紫杉醇的途径上，并破译 羟基和可能的生物合成功能 乙酰化过程； （3）阐明氧烷环的酶促途径 形成； （4）确定晚期的顺序和机制 紫杉醇核的C-2和C-13的酰基化； （5）确认 体内研究的途径并描述速率限制步骤 路径。 描述紫杉醇的复杂途径的目标将 可以通过使用T。 Brevifolia和放射性标记的前体的使用。 放射标记的产品 将采用早期酶促反应来指导 隔离和鉴定相应的中间体 出租车提取物，从中标记为成功酶促的底物 将准备转换。 使用14CO2的体内研究 然后，完整树苗中的标记将用于确认途径，并将其标记 结合同位素稀释分析，以确定速率 限制步骤。 速率限制催化剂和其他酶 与半合成努力相关的途径，从更多 随时可用的紫杉烷代谢物将成为更详细的重点 研究是遗传和分子方法的基础 改善了靶化合物的体内或体外产生。