Structure, Function and Mechanism of Nudix Hydrolases

Nudix 水解酶的结构、功能和机制

• 批准号: 6739079
• 负责人: L. Mario Amzel
• 金额: $ 28.78万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739079
• 关键词: Escherichia coli; Mycobacterium; X ray crystallography; adenosine diphosphate; antibacterial agents; bacterial proteins; chemical kinetics; coenzyme A; electrospray ionization mass spectrometry; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate analog; enzyme substrate complex; peptide chemical synthesis; protein purification; pyrophosphatase; ribose; selenomethionine; site directed mutagenesis

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.

描述（由申请人提供）：nudix酶，磷酸甘氨酸酶，催化核苷酸衍生物中双磷酸链接的水解，其特征在于签名序列GX5EX7REUXEEX2U的存在。从原核生物到真核生物的物种中存在，其假定的作用是控制有毒代谢物和信号分子的细胞水平。 Nudix家族的水解酶家族可以根据其底物特异性，催化活性和表型分为家族。主要的亚家族是：a）Mutt家族，该家族通过消除将8-氧化-DGTP掺入DNA来防止突变； b）与细菌中易尿酸盐耐药性相关的ADP-核糖焦磷酸酶； c）APNA水解酶，其中AP5A水解酶是E. coil K1的侵袭性决定因素； d）辅酶A焦磷酸水解酶和NADH水解酶，尚未确定表型。 Nudix签名序列是针对焦磷酸水解量身定制的循环螺旋 - 环折叠，促进了催化中心，而赋予底物特异性的残基发生在从Nudix基序中取出的序列的区域中发生。催化和识别作用的这种分离为Nudix超级水解酶提供了多功能性。我们建议1）确定哺乳动物和（迈co）细菌量的关键残基的差异，以指导新型抗菌剂的发展； 2）确定辅酶A焦磷酸酶的底物特异性和催化活性的决定因素，辅酶A（一种新近鉴定的Nudix酶亚科）的决定因素； 3）确定E. coil蛋白YGDP的机理和特异性，这是E. coil K1的侵袭能力所必需的NUDIX水解酶。为了解决这些问题，我们设计了特定的实验，这些实验将使用动力学测量，底物模拟设计，机械研究，突变研究和X射线晶体学。拟议的研究将洞悉迅速扩展的Nudix酶家族中每个酶的细节以及基本信息。