MECHANISMS OF BIOSYNTHESIS OF BRANCHED-CHAIN SUGARS

支链糖生物合成机制

• 批准号: 2734803
• 负责人: HUNG-WEN LIU
• 金额: $ 17.21万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734803
• 关键词: antibiotics; bacterial polysaccharides; bacterial proteins; carbohydrate biosynthesis; chemical structure function; drug design /synthesis /production; enzyme mechanism; enzyme structure; enzyme substrate; microorganism metabolism; molecular cloning; protein purification; transfection

DESCRIPTION: The branched-chain sugars are an important class of carbohydrates found widely in nature. They are formally derived from common sugars by replacement of either a hydrogen or a hydroxyl group on a secondary carbon atom with an alkyl side chain. Such a substitution generally causes a critical alteration of the biological function of the resulting sugar, and also induces a fundamental change in its metabolism. Particularly notable are the branched-chain sugars found in many antibiotics in which these unusual sugars play an indispensable role in conferring optimal activity on these natural products. Although the biological importance of branched-chain sugars is well recognized, little is known about the biosynthesis pathways leading to the formation of two branched-chain sugars, yersiniose A and dihydrostreptose. Yersiniose A is an immunodominant sugar found in the lipopolysaccharide (LPS) of Yersinia pseudotuberculosis VI, and dihydrostreptose is a structural component of streptomycin antibiotics isolated from Streptomyces griseus. Emphasis will be placed on the mechanistic studies of enzymes catalyzing the branched-chain construction steps in both cases. The proposed experiments include: 1) to clone the genes encoding the target enzymes and express these genes in E. coli to give catalytically active proteins; 2) to develop appropriate methods to assay the activity of the target enzymes; 3) to purify the target enzymes and characterize their physical and biochemical properties; 4) to prepare alternative substrates as mechanistic probe to study these enzymatic reactions; 5) to elucidate the detailed reaction mechanisms of these enzymes. An understanding of the molecular basis of the biosynthetic formation of these branched-chain sugars will not only aid in delineating how chemical transformations are effected by enzymes catalyzing these conversions, but will also provide invaluable knowledge for designing approaches to control and/or mimic their production and biological activities. Since the significance of sugar residues in determining the biological activity of the antibiotics has been well established, and some of the glycosyl transferases involved in the biosynthesis of antibiotics have been shown to have somewhat relaxed substrate specificity, it is expected that the new insights gained from these studies will also lay groundwork for gene transfer experiments to produce novel or hybrid antibiotics.

描述：支链糖是一类重要的糖。 自然界中广泛存在的碳水化合物。 它们在形式上源自共同的 通过取代糖上的氢或羟基 具有烷基侧链的仲碳原子。 这样的替换 通常会导致生物功能的重大改变 产生的糖，也引起其新陈代谢的根本变化。 特别值得注意的是许多抗生素中发现的支链糖 其中这些不寻常的糖在赋予 这些天然产物的最佳活性。 虽然生物 支链糖的重要性已广为人知，但知之甚少 关于导致两种物质形成的生物合成途径 支链糖、耶尔森糖 A 和二氢链霉糖。 耶尔森氏菌A是 耶尔森氏菌脂多糖 (LPS) 中发现的免疫显性糖 假结核VI，二氢链霉是其结构成分 从灰色链霉菌中分离出的链霉素抗生素。 强调将 致力于酶催化机理研究 两种情况下的支链构建步骤。 拟议的实验 包括：1）克隆编码目标酶的基因并表达 这些基因在大肠杆菌中产生具有催化活性的蛋白质； 2）开发 测定目标酶活性的适当方法； 3）到 纯化目标酶并表征其物理和生化 特性; 4) 准备替代基板作为机械探针 研究这些酶促反应； 5) 阐明详细反应 这些酶的作用机制。 对分子基础的理解 这些支链糖的生物合成形成不仅有助于 描述酶催化如何影响化学转化 这些转换，但也将为设计提供宝贵的知识 控制和/或模仿其生产和生物的方法 活动。 由于糖残基在确定 抗生素的生物活性已得到充分证实，并且一些 参与抗生素生物合成的糖基转移酶 已被证明具有稍微宽松的底物特异性， 预计从这些研究中获得的新见解也将奠定 基因转移实验的基础，以产生新的或杂交的 抗生素。