Structure, Function and Mechanism of Nudix Hydrolases

描述(由申请人提供)：Nudex酶是催化核苷酸衍生物中的二磷酸连接的水解酶，其特征是存在签名序列GX5EX7REUXEEX2U。它们存在于从原核生物到真核生物的各种物种中，其假定的作用是控制有毒代谢物和信号分子的细胞水平。根据底物专一性、催化活性和表型，Nudex水解酶家族可以划分为不同的家族。主要的亚家族是：a)MUT家族，它通过消除8-oxo-dGTP在DNA中的掺入来防止突变；b)ADP-核糖焦磷酸酶，与细菌对亚硫酸盐的抗性有关；c)Apna水解酶，其中的Ap5A水解酶是E.coil K1的侵袭性决定因素；以及d)辅酶A焦磷水解酶和NADH水解酶，其表型尚未确定。 Nudex签名序列为焦磷酸水解量身定做的环-螺旋-环折叠，贡献了催化中心，而赋予底物专一性的残基出现在从Nudex基序移除的序列区域。这种催化和识别作用的分离为Nudex超家族水解酶提供了多功能性。我们建议1)确定哺乳动物和(Myco)细菌ADPRase之间关键残基的差异，以指导新型抗菌药物的开发；2)确定新近发现的Nudex酶亚家族辅酶A焦磷酸酶底物专一性和催化活性的决定因素；以及3)确定E.coil蛋白Ygdp的机制和特异性，它是E.coil K1侵袭能力所必需的Nudex水解酶。为了解决这些问题，我们设计了具体的实验，这些实验将使用动力学测量、底物模拟设计、机理研究、突变研究和X射线结晶学。拟议的研究将深入了解每一种酶家族的细节，以及迅速扩大的Nudex酶家族的基本信息。

DESCRIPTION (provided by applicant): Nudix enzymes, phosphoanhydrases that catalyze the hydrolysis of a diphosphate linkage in nucleotide derivative, are characterized by the presence of the signature sequence GX5EX7REUXEEX2U. Present in species ranging from prokaryotes to eukaryotes, their postulated role is to control the cellular levels of toxic metabolites and signaling molecules. The Nudix family of hydrolases can be divided into families based on their substrate specificity, catalytic activity and phenotype. The major subfamilies are: a) the MutT family, which prevents mutations by eliminating the incorporation of 8-oxo-dGTP into DNA; b) the ADP-ribose pyrophosphatases, which have been associated with tellurite resistance in bacteria; c) the ApnA hydrolases, of which an Ap5A hydrolase is an invasiveness determinant of E. coil K1; and d) the Coenzyme A pyrophosphohydrolases and NADH hydrolases for which no phenotype has yet been determined. The Nudix signature sequence, which folds as a loop-helix-loop tailored for pyrophosphate hydrolysis, contributes the catalytic center, while residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix super family of hydrolases. We propose to 1) identify differences in key residues between mammalian and (myco) bacterial ADPRases that will guide the development of novel antibacterial agents; 2) identify the determinants of substrate specificity and catalytic activity of Coenzyme A pyrophosphatases, a newly identified subfamily of Nudix enzymes; and 3) determine the mechanism and the specificity of E. coil protein Ygdp, a Nudix hydrolase necessary for the invasion competence of E. coil K1. To address these questions, we designed specific experiments that will use kinetic measurements, substrate analog design, mechanistic studies, mutational studies and x-ray crystallography. The proposed research will provide insight into the details of each family of enzymes as well as basic information in the rapidly expanding family of Nudix enzymes.