Structure and Mechanism in the Tautomerase Superfamily

互变异构酶超家族的结构和机制

• 批准号: 6876691
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6876691
• 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）：互变异酶超家族由 基于β-alpha-beta主题的结构同源酶，其成员 使用Pro-I作为互变体和异构反应中的一般基础。 这项研究的长期目标是确定分子和 互变异酶超家族中催化和特异性的结构基础。 这项研究对我们对基本酶促的理解有影响 反应和酶的进化。此外，这些研究将有助于 在确定各种致病性的代谢能力范围 有机体，有可能导致新药的开发，并促进 生物修复工作。 本申请的重点将是代表成员 4-恶性核苷酸互变异酶（4-OT）家族。这些酶的大小从 每个单体61-79氨基酸，均具有氨基末端脯氨酸。这 首席研究员小组最近发现了结构性和 这个家庭的机械多样性。这种多样性表明自然使用了 这些简短的序列（编码简单的beta-alpha-beta主题）以创建新的 结构和活动。主要目的是确定 1）3-氯丙烯酸脱脂酶的机理和结构，可能 使用pro-1激活水进行添加反应，2）丙二酸酯半甲醛 脱羧酶，可以在Schiff基础机制中使用Pro-1来促进 脱羧，3）互变异酶，其水平异构酶和 脱核酶活性，4）二聚体4-OT同源物，5）紧密 具有互变异酶和异构酶活性但缺乏的相关同源物 在父构件中发现的保守活性位点残基（Pro-1除外），4-OT。 最后，提出了实验以改善低级活动和 通过理性设计来操纵低聚物状态。这些研究将提供 更好地理解每个酶的结构/功能关系。一个 比较策略和主动地点结构将提供签名 对于每种酶活性，阐明了这些活动的发展方式， 协助分配功能。结果，基本原则 将在此系统中使用，以便自然的过程可以 模仿使用beta-a-beta图案创建新的活动和结构。