Structure, Function and Mechanism of Nudix Hydrolases

Nudix 水解酶的结构、功能和机制

• 批准号: 7060040
• 负责人: L. Mario Amzel
• 金额: $ 28.1万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060040
• 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，即催化核苷酸衍生物中二磷酸键水解的磷酸酐酶，其特征在于存在特征序列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。 Nudix 水解酶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，它通过消除 8-oxo-dGTP 掺入 DNA 来防止突变； b) ADP-核糖焦磷酸酶，其与细菌的亚碲酸盐抗性有关； c) ApnA水解酶，其中Ap5A水解酶是大肠杆菌K1的侵袭性决定因素； d) 尚未确定表型的辅酶 A 焦磷酸水解酶和 NADH 水解酶。 Nudix 特征序列折叠为专为焦磷酸水解而设计的环-螺旋-环，提供催化中心，而赋予底物特异性的残基出现在从 Nudix 基序中去除的序列区域中。这种催化和识别作用的分离为 Nudix 水解酶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 ADPRase 之间关键残基的差异，这将指导新型抗菌剂的开发； 2) 确定辅酶 A 焦磷酸酶（新鉴定的 Nudix 酶亚家族）底物特异性和催化活性的决定因素； 3)确定大肠杆菌蛋白Ygdp的机制和特异性，Ygdp是大肠杆菌K1入侵能力所必需的Nudix水解酶。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、基质模拟设计、机理研究、突变研究和 X 射线晶体学。拟议的研究将深入了解每个酶家族的详细信息以及快速扩展的 Nudix 酶家族的基本信息。

项目成果

Structures and mechanisms of Nudix hydrolases.

Nudix 水解酶的结构和机制。

• 发表时间: 2005-01-01
• 影响因子: 3.9
• 作者: Mildvan, A S;Xia, Z;Azurmendi, H F;Saraswat, V;Legler, P M;Massiah, M A;Gabelli, S B;Bianchet, M A;Kang, L;Amzel, L M
• 通讯作者: Amzel, L M

X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction.

GDP-甘露糖甘露糖基水解酶反应催化循环的 X 射线、NMR 和突变研究。

• 发表时间: 2006-09-26
• 影响因子: 2.9
• 作者: Gabelli, Sandra B;Azurmendi, Hugo F;Bianchet, Mario A;Amzel, L Mario;Mildvan, Albert S
• 通讯作者: Mildvan, Albert S

Structural and thermodynamic insights into the recognition of native proteins by anti-peptide antibodies.

抗肽抗体识别天然蛋白质的结构和热力学见解。

• 发表时间: 2013-06-12
• 影响因子: 5.6
• 作者: Armstrong, AnthonyA;Hildreth, James E K;Amzel, L Mario
• 通讯作者: Amzel, L Mario

Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis.

大肠杆菌二氢新蝶呤三磷酸焦磷酸酶的结构和功能：一种参与叶酸生物合成的 Nudix 酶。

• 发表时间: 2007-08
• 作者: Gabelli, Sandra B;Bianchet, Mario A;Xu, WenLian;Dunn, Christopher A;Niu, Zhi;Amzel, L Mario;Bessman, Maurice J
• 通讯作者: Bessman, Maurice J

Identification of Bdellovibrio bacteriovorus HD100 Bd0714 as a Nudix dGTPase.

将噬菌蛭弧菌 HD100 Bd0714 鉴定为 Nudix dGTPase。

• 发表时间: 2008-12
• 影响因子: 3.2
• 作者: Steyert, Susan R;Messing, Simon A J;Amzel, L Mario;Gabelli, Sandra B;Piñeiro, Silvia A
• 通讯作者: Piñeiro, Silvia A