Amidohydrolase Superfamiily

酰胺水解酶超家族

• 批准号: 6854961
• 负责人: Frank M. Raushel
• 金额: $ 28.32万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6854961
• 关键词: X ray crystallography; active sites; aminohydrolases; biochemical evolution; bioinformatics; chemical bond; chemical reaction; combinatorial chemistry; enzyme activity; enzyme structure; enzyme substrate; high throughput technology; hydrolysis; method development; protein purification; protein structure function

DESCRIPTION (provided by applicant) The long-term objective for the research described within this portion of the program project is aimed toward the development of general and comprehensive methodologies for the determination of reaction and substrate specificities for proteins of unknown function. This goal will be pursued by concentrating on the elucidation of the substrate and reaction profiles for the entire ensemble of enzymes within the amidohydrolase superfamily. This superfamily of enzymes has been shown to catalyze the hydrolysis of P-O, P-S, P-F, P-C, C-N, C-O, and C-CI bonds in addition to the cleavage of C-C bonds. The structural hallmark for this superfamily of enzymes is an active site at the C-terminal end of an (beta/alpha)8-barrel protein 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. Prominent members of this family of enzymes include dihydroorotase, urease, phosphotriesterase, and adenosine deaminase. 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. Bioinformatic analyses have identified more than 2,000 members of this superfamily within the 1 million proteins sequenced to date from all organisms. Moreover, in excess of thirty different chemical reactions have been identified thus far for members of this superfamily of enzymes and preliminary evidence suggests that more than 100 additional reactions remain to be elucidated. The specific chemical reactions catalyzed by members of this superfamily with unknown catalytic functions will be experimentally determined by direct biochemical assessment of substrate libraries generated with the aid of high throughput computational docking in conjunction with high resolution x-ray crystallography. These studies will complement the cloning, expression, and purification of specific members of the Superfamily where the biochemical reaction profile is currently unknown. In order to enhance the modeling of the amidohydrolase active sites and docking protocols, representative examples for all of the known isofunctional members of this enzyme superfamily will be structurally characterized by x-ray crystallography. The concerted and synergistic application of biochemical, computational, and structural methodologies described in this application will amplify the evolutionary diversity of the reactions catalyzed by members of the amidohydrolase superfamily and, more importantly, provide general and practical approaches for the assignment of function to proteins of unknown substrate and reaction specificity.

描述（由申请人提供） 该计划项目的这一部分中描述的研究的长期目标是开发通用和综合的方法来确定未知功能蛋白质的反应和底物特异性。将通过集中阐明酰胺水解酶超家族内整个酶群的底物和反应谱来实现这一目标。该酶超家族已被证明除了 C-C 键的裂解外，还可催化 P-O、P-S、P-F、P-C、C-N、C-O 和 C-CI 键的水解。该酶超家族的结构特征是 (β/α)8 桶蛋白 C 末端的活性位点，该蛋白含有单核或双核金属中心，其主要但并非唯一的功能是激活溶剂水对亲电官能团的亲核攻击。该酶家族的重要成员包括二氢乳清酶、脲酶、磷酸三酯酶和腺苷脱氨酶。该酶超家族中包含的底物和反应多样性将为从现有结构模板进化和发展新型酶活性的分子机制提供独特的见解。生物信息学分析发现了更多 迄今为止，已对 100 万个蛋白质进行了测序，其中包含该超家族的 2,000 多个成员 有机体。此外，迄今为止，该酶超家族成员已鉴定出超过 30 种不同的化学反应，初步证据表明还有 100 多种其他反应有待阐明。该超家族成员催化功能未知的特定化学反应将通过对借助高通量计算对接与高分辨率 X 射线晶体学生成的底物库进行直接生化评估来实验确定。这些研究将补充目前生化反应谱未知的超家族特定成员的克隆、表达和纯化。为了增强酰胺水解酶活性位点和对接方案的建模，该酶超家族所有已知同功能成员的代表性实例将通过 X 射线晶体学进行结构表征。本申请中描述的生物化学、计算和结构方法的协同和协同应用将放大由酰胺水解酶超家族成员催化的反应的进化多样性，更重要的是，为将功能分配给酰胺水解酶超家族的蛋白质提供通用和实用的方法。未知的底物和反应特异性。