Structure and Mechanism in the Tautomerase Superfamily

描述（由申请人提供）：互变异构酶超家族由以下组成： 基于β-α-β基序的结构同源酶，其成员 在互变异构化和异构化反应中使用Pro-i作为通用碱。 这项研究的长期目标是确定分子和 在互变异构酶超家族中催化和特异性的结构基础。 这项研究对我们理解基本的酶促反应具有启示作用。 反应和酶的进化。此外，这些研究将有助于 在确定各种病原体的代谢能力范围时， 生物，可能导致新药的开发，并促进 生物补救措施。 本申请的重点将是代表成员的 4-异巴豆酸互变异构酶（4-OT）家族。这些酶的大小范围从 每个单体61-79个氨基酸，并且都具有氨基末端脯氨酸。的 首席研究员的小组最近发现， 机械的多样性。这种多样性表明，大自然使用 这些短序列（编码简单的β-α-β基序）， 结构和活动。主要的具体目标将是确定 1）3-氯丙烯酸脱卤酶的机理和结构， 用Pro-1活化水进行加成反应，2）丙二酸半醛 脱羧酶，其可以在席夫碱机制中使用Pro-1以促进 3）互变异构酶，其具有低水平的异构酶，和 脱卤酶活性，4）二聚体4-OT同源物，和5）两个密切 具有互变异构酶和异构酶活性但缺乏 在亲本成员4-OT中发现的保守活性位点残基（Pro-1除外）。 最后，提出了改进低层活动的实验， 通过合理的设计来操纵低聚物状态。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 通过对策略和活性位点结构的比较， 对于每种酶活性，阐明这些活性是如何进化的， 协助分配职能。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 通过模仿来创造新的活性和结构。

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.