Amidohydrolase Superfamiily

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旨在开发用于确定未知功能蛋白质的反应和底物特异性的通用和综合方法。这一目标将通过集中在阐明的底物和反应概况的整个合奏酶内的酰胺水解酶超家族。除了C-C键的裂解之外，该酶超家族已显示催化P-O、P-S、P-F、P-C、C-N、C-O和C-Cl键的水解。该酶超家族的结构标志是在（β/α）8桶蛋白的C-末端的活性位点，其含有单核或双核金属中心，其主要但不排他地起作用以活化溶剂水用于亲电子官能团的亲核攻击。该酶家族的主要成员包括二氢乳清酸酶、尿素酶、磷酸三酯酶和腺苷脱氨酶。该酶超家族中所含的底物和反应多样性将为从现有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， 在迄今为止测序的100万个蛋白质中， 有机体此外，到目前为止，已经确定了超过30种不同的化学反应，初步证据表明，超过100个其他反应仍有待阐明。由具有未知催化功能的这个超家族的成员催化的特定化学反应将通过对借助于高通量计算对接结合高分辨率X射线晶体学产生的底物库的直接生物化学评估来实验确定。这些研究将补充克隆，表达和纯化的特定成员的超家族的生化反应谱是目前未知的。为了增强酰胺水解酶活性位点和对接方案的建模，该酶超家族的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。在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的成员催化的反应的进化多样性，并且更重要的是，为未知底物和反应特异性的蛋白质的功能分配提供一般和实用的方法。

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.