MECHANISMS OF BIOSYNTHESIS OF BRANCHED-CHAIN SUGARS

支链糖生物合成机制

• 批准号: 2444902
• 负责人: HUNG-WEN LIU
• 金额: $ 15.45万
• 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/2444902
• 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; 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：分支链糖是重要类 自然界中发现的碳水化合物广泛。 它们正式衍生自共同 糖通过在A上取代氢或羟基 具有烷基侧链的次级碳原子。 这样的替代 通常会导致对生物学功能的严重改变 产生的糖，还引起其新陈代谢的根本变化。 在许多抗生素中发现的分支链糖特别值得注意 其中这些不寻常的糖在授予中起着必不可少的作用 这些天然产品的最佳活性。 虽然生物学 分支链糖的重要性已被众所周知，知之甚少 关于导致两个形成的生物合成途径 分支链糖，yersiniose a和二氢诱变。 yersiniose a是 在耶尔森氏菌的脂多糖（LPS）中发现的免疫主导糖 假结论VI和二氢诱变是的结构成分 从谷物链霉菌分离的链霉素抗生素。 强调会 放置在催化酶的机械研究上 在两种情况下，分支链建筑步骤。 提出的实验 包括：1）克隆编码靶酶并表达的基因 这些基因在大肠杆菌中产生催化活性蛋白。 2）开发 适当的方法来分析靶酶的活性； 3）到 净化靶酶并表征其物理和生化 特性; 4）准备替代底物作为机械探针 研究这些酶促反应； 5）阐明详细反应 这些酶的机制。 对分子基础的理解 这些分支链糖的生物合成形成不仅有助于 描述如何通过催化酶实现化学转化 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学的方法 活动。 由于糖残基在确定 抗生素的生物学活性已经建立得很好，有些 参与抗生素生物合成的糖基转移酶 已证明具有放松的底物特异性，这是 预计从这些研究中获得的新见解也将是 基因转移实验的基础工作，以产生新颖或杂种 抗生素。