METALLO BETA LACTAMASE FROM X MALTOPHILIA

来自 X Maltophilia 的 METALLO β 内酰胺酶

• 批准号: 6124376
• 负责人: MICHAEL W CROWDER
• 金额: $ 10.48万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124376
• 关键词: Pseudomonadaceae; X ray spectrometry; active sites; beta lactam antibiotic; beta lactamase; cephalosporins; chemical kinetics; drug design /synthesis /production; drug resistance; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; mass spectrometry; mathematical model; nuclear magnetic resonance spectroscopy; penicillins; site directed mutagenesis; stop flow technique; structural biology; zinc; Pseudomonadaceae; X ray spectrometry; active sites; beta lactam antibiotic; beta lactamase; cephalosporins; chemical kinetics; drug design /synthesis /production; drug resistance; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; mass spectrometry; mathematical model; nuclear magnetic resonance spectroscopy; penicillins; site directed mutagenesis; stop flow technique; structural biology; zinc

Enzymes that hydrolyze beta-lactams and cephalosporin antibiotics are called beta-lactamases, and their production by bacteria is the most common way that bacteria become antibiotic-resistant. The Zn(II)- containing beta-lactamases constitute an ever-growing and troubling class of beta-lactamases which contain 2 moles of Zn(II) per mole of enzyme, hydrolyze all known penicillin and cephalosporin antibiotics, are not inhibited by clavulanic acid, and have no known inhibitor of activity. The long-term objective of the proposed research is the rational design and preparation of irreversible inhibitors of the Zn(II)-containing beta-lactamases. Given the apparent structural and mechanistic differences among the metallo-beta-lactamases, this objective can only be realized after detailed characterization of a member from each distinct subclass of metallo-beta-lactamases. Similarities between the enzymes can then be identified and exploited for the rational design of an inhibitor. This proposal involves the study of the structurally-distinct, biomedically-important beta- lactamase (L1) from X maltophilia. The specific aims of this proposed research are: (1) detailed structural characterization of the metal binding sites of metal-substituted forms of the X. maltophilia beta- lactamase using spectroscopic, mutagenesis, and binding studies, (2) elucidation of the mode of action of the enzyme with several penicillin and cephalosporin antibiotics using steady-state and pre-steady state kinetics studies, (3) identification of the overall reaction mechanism used by Ll and of any stable reaction intermediates using mathematical simulations of kinetics rate profiles and results from biochemical studies, (4) integration of structural and mechanistic results to identify key aspects of the enzyme that can be targeted for the development of novel reaction- or structure-based inhibitors, and (5) examination of the possible inhibition properties of compounds, when once hydrolyzed produce reactive, exocyclic methylene groups. It is hoped that this novel integration of kinetics, biochemical, simulations, and spectroscopic studies can be used as a general strategy for the preparation of new antimicrobial agents and a better way to combat the ever-increasing prevalence of antibiotic resistance in bacteria.

水解β-内酰胺和头孢菌素抗生素的酶是 称为β-内酰胺酶，细菌的产生最多 细菌变得抗生素的常见方式。 Zn（ii） - 含有β-内酰胺酶构成了不断增长且令人不安的 β-内酰胺酶一类，每摩尔的锌（II）含2摩尔 酶，水解所有已知的青霉素和头孢菌素抗生素， 不受克拉维拉酸抑制，并且没有已知的抑制剂 活动。 拟议研究的长期目标是 理性的设计和制备不可逆抑制剂 Zn（II） - 含β-内酰胺酶。考虑到明显的结构和 金属β-内酰胺酶之间的机械差异，这 只有在详细表征的表征之后才能实现目标 来自金属β-内酰胺酶的每个不同亚类的成员。 然后可以识别和利用酶之间的相似性 用于抑制剂的合理设计。 该建议涉及 研究结构存在的生物医学重要β- X麦芽脂糖酶（L1）。 该提议的具体目的 研究是：（1）金属的详细结构表征 X. mathophilia beta-的金属取代形式的结合位点 使用光谱，诱变和结合研究的乳糖酶，（2） 用几种青霉素阐明酶的作用方式 使用稳态和稳态状态的头孢菌素抗生素 动力学研究，（3）鉴定总体反应机理 LL和任何稳定的反应中间体使用数学使用 动力学率曲线和生化结果的模拟 研究，（4）结构和机械结果的整合到 确定可以针对酶的酶的关键方面 开发新的反应或基于结构的抑制剂，（5） 检查化合物可能的抑制特性 一旦水解产生反应性，外生甲基基团。 这是 希望这种动力学，生化，仿真， 光谱研究可以用作一般策略 准备新的抗菌剂，也是一种更好的作战方式 细菌中抗生素耐药性的不断增长。