Deciphering Enzyme Specificity: Amidohydrolase Superfamily

破译酶的特异性：酰胺水解酶超家族

基本信息

批准号：
7743893
负责人：
Frank M. Raushel
金额：
$ 30.6万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7743893
关键词：
Active Sites Amides Amidohydrolases Amino Acid Sequence Amino Acids Biochemical Biological Assay C-terminal Chemicals Code Collaborations Complement Deamination Decarboxylation Detection Development Dihydroorotase Docking Enzymes Esters Evolution Funding Genes Genomics Goals Homology Modeling Hydration status Hydrolysis Instruction Laboratories Lactones Libraries Ligand Binding Metabolic Metabolic Pathway Metals Methodology Methods Modeling Molecular Mononuclear New York Nucleic Acids Operon Organophosphates Peptide Sequence Determination Phosphoric Triester Hydrolases Principal Investigator Proteins Protocols documentation Reaction Research Resolution Roentgen Rays Screening procedure Solvents Specificity Structure Substrate Specificity System Therapeutic Intervention Urease Water adenosine deaminase combinatorial enzyme structure forging functional group insight member novel programs protein structure small molecule small molecule libraries structural genomics

项目摘要

The long-term objective for the research described in this application is directed at a comprehensive understanding of the physical and chemical parameters that relate protein structure and substrate recognition in enzyme-catalyzed reactions. This objective is aimed toward the development of general methodologies and novel protocols for the determination of reaction and substrate specificities for enzymes of unknown function. A critical assessment of the functional annotations forthe more than four million genes that have been sequenced to date suggests that approximately one-third of the encoded proteins have an uncertain, unknown, or /ncorrecf functional assignment. This observation suggests that a significant fraction of the metabolic diversity remains to be properly characterized. Toward this end we will utilize computational docking of high energy intermediates to models of the active sites for enzymes of unknown function to identify the most probable substrates. These efforts will be complemented by the synthesis and screening of chemical libraries and the abstraction of further metabolic information from operon and genomic context. This goal will be pursued by concentrating on the elucidation of the substrate and reaction profiles forthe entire ensemble of enzymes within the amidohydrolase superfamily. The amidohydrolase superfamily is a group of enzymes which has a substantial substrate diversity embedded within active sites that are forged from a (p/a)8-barrel structural fold. Over 6,000 unique protein sequences have been identified as members of the amidohydrolase superfamily. Members of this superfamily have been shown to catalyze the hydrolysis of amides, lactones and organophosphate esters, in addition to decarboxylation, hydration, and isomerization reactions. However, a substantial fraction of the members of this broad superfamily have an ambiguous substrate and reaction specificity that remains to be unraveled. Members of this superfamily of enzymes include dihydroorotase, urease, and adenosine deaminase. The hallmark for this particular superfamily of enzymes is an active site at the C-terminal end of a (p/a)8-barrel structural domain that contains a mononuclear or binuclear metal center that functions predominantly, but not exclusively, to activate solvent water for nucleophilic attack on electrophilic functional groups. The substrate and reaction diversity contained within this enzyme superfamily will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities from existing structural templates. RELEVANCE (See instructions); The overall objective of this application is directed towards the development of novel and general methods for the elucidation of function for enzymes with unknown substrates. These efforts will unveil new metabolic transformations and identify new targets for therapeutic intervention.

本申请中描述的研究的长期目标是针对全面的了解与蛋白质结构和底物相关的物理和化学参数酶催化反应中的识别。这一目标旨在促进一般性的发展用于确定酶的反应和底物特异性的方法和新方案未知功能。对超过四百万个基因的功能注释进行严格评估迄今为止已测序的结果表明，大约三分之一的编码蛋白质具有不确定、未知或 /ncorrecf 功能分配。这一观察结果表明，很大一部分代谢多样性的特征仍有待适当表征。为此，我们将利用计算将高能中间体与未知功能酶的活性位点模型对接确定最可能的底物。这些努力将得到合成和筛选的补充化学文库以及从操纵子和基因组背景中提取进一步的代谢信息。这一目标将通过集中阐明底物和反应曲线来实现。酰胺水解酶超家族中的整个酶系。酰胺水解酶超家族是一组具有大量底物多样性的酶，嵌入在锻造的活性位点中来自 (p/a)8 桶结构折叠。超过 6,000 个独特的蛋白质序列已被鉴定为酰胺水解酶超家族。该超家族的成员已被证明能够催化水解酰胺、内酯和有机磷酸酯，以及脱羧、水合和异构化反应。然而，这个广泛的超家族的大部分成员都有一个模糊的概念底物和反应特异性仍有待阐明。该酶超家族的成员包括二氢乳清酶、脲酶和腺苷脱氨酶。这个特殊超家族的标志酶是位于 (p/a)8 桶结构域 C 末端的活性位点，该结构域包含单核或双核金属中心，主要但不限于活化溶剂水对亲电官能团进行亲核攻击。底物和反应多样性该酶超家族中所包含的内容将为了解该酶的分子机制提供独特的见解。从现有结构模板进化和开发新的酶活性。相关性（参见说明）；该应用程序的总体目标是开发新颖和通用的阐明具有未知底物的酶的功能的方法。这些努力将揭开新面纱代谢转变并确定治疗干预的新目标。