Structure, Function and Mechanism of Nudix Hydrolases

描述（由申请人提供）：核酸酶，即催化核苷酸衍生物中二磷酸键水解的磷酸酸酐酶，其特征在于存在特征序列GX 5EX 7 REUXEEX 2U。它们存在于从原核生物到真核生物的各个物种中，其假定作用是控制有毒代谢物和信号分子的细胞水平。水解酶的Nutrition家族可根据其底物特异性、催化活性和表型分为多个家族。主要的亚家族是：a）MutT家族，其通过消除8-氧代-dGTP掺入DNA来防止突变; B）ADP-核糖焦磷酸酶，其与细菌中的亚碲酸盐抗性相关;螺旋K1;和d）辅酶A焦磷酸水解酶和NADH水解酶，其表型尚未确定。 Nutrient签名序列，其折叠为针对焦磷酸水解定制的环-螺旋-环，贡献了催化中心，而赋予底物特异性的残基出现在从Nutrient基序移除的序列区域中。这种催化和识别作用的分离为水解酶的Nutrium超家族提供了多功能性。我们建议1）确定哺乳动物和（真菌）细菌ADPRases之间的关键残基的差异，这将指导新型抗菌剂的开发; 2）确定辅酶A焦磷酸酶（一种新发现的营养素酶亚家族）的底物特异性和催化活性的决定因素; 3）确定E. coil蛋白Ygdp是大肠杆菌入侵能力所必需的一种营养素水解酶。线圈K1。为了解决这些问题，我们设计了具体的实验，将使用动力学测量，底物模拟设计，机理研究，突变研究和X射线晶体学。拟议的研究将提供深入了解每个酶家族的细节以及快速扩展的营养素酶家族的基本信息。

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.