Structure and Mechanism in the Tautomerase Superfamily

描述(申请人提供)：互变擦除酶超家族包括 基于其成员的β-α-β基序的结构同源酶 在互变异构化和异构化反应中使用Pro-I作为总碱。 这项研究的长期目标是确定分子和 互变清除酶超家族催化和特异性的结构基础。 这项研究对我们理解基础酶有一定的启示。 反应和酶的进化。此外，这些研究将有助于 在确定不同病原体的代谢能力范围时 生物体，潜在地导致新药的开发，并促进 生物修复的努力。 此应用程序的焦点将是 4-草酰基转移酶(4-OT)家族。这些酶的大小从 每个单体有61-79个氨基酸，都有一个氨基末端的脯氨酸。这个 首席调查组最近发现了结构和 这个家庭中的机械多样性。这种多样性表明，大自然曾利用 这些短序列(编码一个简单的β-α-β基序)创建新的 结构和活动。主要的具体目标将是确定 1)3-氯丙烯酸脱卤酶的作用机理和结构 使用Pro-1活化水进行加成反应，2)丙二酸半醛 脱羧酶，它可以利用Pro-1中的席夫碱机制来促进 脱羧基，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同系物，以及5)两个紧密的 具有互换消除酶和异构酶活性但缺乏 在亲本成员4-OT中发现保守的活性中心残基(Pro-1除外)。 最后，提出了改进低层次活动的实验方案，并提出了改进方案。 通过合理的设计来控制齐聚物的状态。这些研究将提供一个 更好地理解每种酶的结构/功能关系。一个 战略和活动站点结构的比较将提供签名 对于每一种酶活性，阐明这些活性是如何演变的，以及 协助完成职能的分配。因此，基本原则 在这个系统中使用的将被识别，以便自然的过程可以 模仿使用Beta-a-beta主题创建新的活动和结构。

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.