METALLO BETA LACTAMASE FROM X MALTOPHILIA

来自 X Maltophilia 的 METALLO β 内酰胺酶

• 批准号: 2469460
• 负责人: MICHAEL W CROWDER
• 金额: $ 10.83万
• 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/2469460
• 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

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-内酰胺类和头孢菌素类抗生素的酶是 称为β-内酰胺酶，细菌产生的β-内酰胺酶是最常见的 这是细菌产生抗药性的常见方式。Zn（II）- 含有β-内酰胺酶的细菌， 每摩尔β-内酰胺酶含有2摩尔Zn（II）的β-内酰胺酶 酶，水解所有已知的青霉素和头孢菌素抗生素， 不受克拉维酸的抑制，也没有已知的 活动 拟议研究的长期目标是 不可逆抑制剂的合理设计和制备 含锌β-内酰胺酶。鉴于明显的结构和 金属β-内酰胺酶之间的机制差异，这 只有在详细描述了一个 来自金属β-内酰胺酶的每个不同亚类的成员。 酶之间的相似性可以被识别和利用 用于抑制剂的合理设计。 该提案涉及 研究结构独特，生物医学重要的β- 嗜麦芽窄食单胞菌的内酰胺酶（L1）。 提出的具体目标 主要研究内容有：（1）金属的详细结构表征 X的金属取代形式的结合位点。嗜麦芽窄食β- 使用光谱、诱变和结合研究的内酰胺酶，（2） 阐明酶与几种青霉素的作用方式 和头孢菌素类抗生素使用稳态和预稳态 动力学研究;（3）反应机理的确定 和任何稳定的反应中间体使用数学 模拟动力学速率曲线和生化反应的结果 研究，（4）整合结构和机械结果， 确定酶的关键方面，可以针对 开发基于反应或结构的新型抑制剂，以及（5） 检查化合物的可能抑制特性，当 一旦水解产生反应性的环外亚甲基。 是 希望这种新颖的动力学，生化，模拟， 和光谱研究可以作为一个一般的战略， 新的抗菌剂的制备和更好地防治 细菌中抗生素耐药性的不断增加。