Structure and Mechanism in the Tautomerase Superfamily

互变异构酶超家族的结构和机制

• 批准号: 6463912
• 负责人: CHRISTIAN P. WHITMAN
• 金额: $ 25.67万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6463912
• 关键词: X ray crystallography; bacterial proteins; chemical kinetics; crystallization; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; hydrolase; intermolecular interaction; isomerase; isozymes; microorganism culture; model design /development; molecular cloning; molecular site; nuclear magnetic resonance spectroscopy; physical model; protein engineering; protein purification; site directed mutagenesis; structural biology; tautomer

DESCRIPTION (provided by applicant): The tautomerase superfamily consists of structurally homologous enzymes based on a beta-alpha-beta motif whose members use Pro-i as the general base in tautomerization and isomerization reactions. The long-term goal of this research is to determine the molecular and structural basis for catalysis and specificity in the tautomerase superfamily. This research has implications for our understanding of fundamental enzymatic reactions and the evolution of enzymes. In addition, these studies will assist in determining the range of metabolic capabilities for various pathogenic organisms, potentially leading to the development of new drugs, and facilitate bio-remediation efforts. The focus of this application will be representative members of the 4-oxalocrotonate tautomerase (4-OT) family. These enzymes range in size from 61-79 amino acids per monomer and all have an amino-terminal proline. The principal investigator's group has recently discovered structural and mechanistic diversity in this family. This diversity suggests that Nature used these short sequences (encoding a simple beta-alpha-beta motif) to create new structures and activities. The major specific aims will be to determine the mechanisms and structures for 1) 3-chloroacrylic acid dehalogenase which may use Pro-1 to activate water for an addition reaction, 2) malonate semialdehyde decarboxylase, which may use Pro-1 in a Schiff base mechanism to facilitate decarboxylation, 3) a tautomerase, which has low level isomerase and dehalogenase activities, 4) a dimeric 4-OT homologue, and 5) two closely related homologues that have tautomerase and isomerase activities but lack the conserved active site residues (except Pro-1) found in the parent member, 4-OT. Finally, experiments are proposed to improve the low-level activities and to manipulate the oligomer state by rational design. These studies will provide a better understanding of the structure/function relationships for each enzyme. A comparison of the strategies and active site structures will provide signatures for each enzymatic activity, shed light on how these activities evolved, and assist in the assignment of function. As a result, the underlying principles used in this system will be identified so that Nature's processes could be mimicked to create new activities and structures using the beta-a-beta motif.

描述（由申请人提供）：互变异构酶超家族包括 基于 β-α-β 基序的结构同源酶，其成员 使用Pro-i作为互变异构和异构化反应的通用碱。 这项研究的长期目标是确定分子和 互变异构酶超家族的催化和特异性的结构基础。 这项研究对我们理解基本酶促反应具有重要意义 反应和酶的进化。此外，这些研究将有助于 确定各种病原体的代谢能力范围 有机体，有可能导致新药的开发，并促进 生物修复工作。 该申请的重点将是该协会的代表成员 4-草酰巴豆酸互变异构酶 (4-OT) 家族。这些酶的大小范围为 每个单体有 61-79 个氨基酸，并且全部具有氨基末端脯氨酸。这 首席研究员小组最近发现了结构和 这个家族的机制多样性。这种多样性表明大自然使用 这些短序列（编码一个简单的β-α-β基序）来创建新的 结构和活动。主要的具体目标是确定 1) 3-氯丙烯酸脱卤酶的机制和结构可能 用Pro-1活化水进行加成反应，2）丙二酸半醛 脱羧酶，它可以在希夫碱机制中使用 Pro-1 来促进 脱羧，3）互变异构酶，具有低水平的异构酶和 脱卤酶活性，4) 二聚体 4-OT 同源物，以及 5) 两个密切相关的 具有互变异构酶和异构酶活性但缺乏互变酶和异构酶活性的相关同系物 在亲本成员 4-OT 中发现了保守的活性位点残基（Pro-1 除外）。 最后，提出了实验来改进低级活动并 通过合理设计控制低聚物状态。这些研究将提供 更好地了解每种酶的结构/功能关系。一个 策略和活动站点结构的比较将提供签名 对于每种酶活性，阐明这些活性是如何进化的，并且 协助分配职能。因此，基本原则 该系统中使用的物质将被识别，以便自然的过程可以 模仿使用 beta-a-beta 主题创造新的活动和结构。