Amidohydrolase Superfamiily

描述(由申请人提供) 该计划项目这一部分中描述的研究的长期目标旨在开发确定未知功能蛋白质的反应和底物特异性的一般和全面的方法。这一目标将通过集中于阐明氨基水解酶超家族中整个酶的底物和反应谱来实现。这一超家族的酶除了催化C-C键的断裂外，还能催化P-O、P-S、P-F、P-C、C-N、C-O和C-CI键的水解。这个超家族酶的结构特征是在(β/α)8桶蛋白的C末端有一个活性部位，它包含一个单核或双核金属中心，主要但不是唯一的功能是激活溶剂水，对亲电官能团进行亲核攻击。这一家族的重要成员包括二氢核酸酶、尿素酶、磷酸三酯酶和腺苷脱氨酶。这个酶超家族所包含的底物和反应多样性将为从现有的结构模板进化和开发新的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 这个超家族的2000多个成员，在迄今从所有 有机体。此外，到目前为止，已经为这一超家族的酶成员确定了30多个不同的化学反应，初步证据表明，还有100多个其他反应有待阐明。这一超家族成员催化的特定化学反应具有未知的催化功能，将通过高通量计算对接结合高分辨率X射线结晶学对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。为了加强对氨基水解酶活性部位和对接协议的模拟，将用X射线结晶学对这个酶超家族所有已知的等功能成员的代表性实例进行结构表征。本申请中描述的生化、计算和结构方法的协同和协同应用将放大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，为将功能分配给未知底物和反应特异性的蛋白质提供通用和实用的方法。

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.