Time-dependent structural studies on dinuclear metal ion containing enzymes

含双核金属离子酶的时间依赖性结构研究

• 批准号: 7940321
• 负责人: MICHAEL W CROWDER
• 金额: $ 42.56万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940321
• 关键词: Active Sites; Address; Amides; Anthrax disease; Antibiotic Resistance; Antibiotics; Arylsulfatases; Bacillus anthracis; Bacterial Infections; Binding; Biochemical Reaction; Catalysis; Cessation of life; Clinic; Clinical; Collection; Coupled; Coupling; Development; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Enzymes; Freezing; Future; Generations; Glean; Gluconolactonase; Goals; Hydrolysis; Ions; Klebsiella pneumonia bacterium; Lactamase; Lactams; Metal Binding Site; Metals; Methodology; Motion; Organism; Penicillin Resistance; Positioning Attribute; Pseudomonas aeruginosa; Reaction; Research; Roentgen Rays; Role; Series; Site; Solutions; Spectrum Analysis; Spin Labels; Stenotrophomonas maltophilia; Structure; Techniques; Time; Transfer RNA; X ray spectroscopy; absorption; base; flexibility; inhibitor/antagonist; metalloenzyme; oxidation; phosphoric diester hydrolase; programs; public health relevance; research study

DESCRIPTION (provided by applicant): This proposal describes studies to probe structural changes in an enzyme during the course of its biochemical reaction. We propose to utilize rapid-freeze quench (RFQ) coupled with several spectroscopic techniques to probe the reaction of metallo-?-lactamase (M?L) L1 from Stenotrophomonas maltophilia as substrate binds and is converted into product. Specifically, ... In Specific aim #1, we propose to explore changes in metal site structure as a function of reaction coordinate, coupling rapid-freeze-quench (RFQ) techniques with conventional spectroscopies, including X-ray absorption spectroscopy (XAS), EPR and electron-nuclear double resonance (ENDOR), in the reaction of L1 with Zn and/or Co at the active site with a ?-lactam substrate. In Specific aim #2, we will examine solution dynamics of a position-conserved flexible loop that covers the metal site during turnover, through CW EPR studies of Co-containing derivatives of L1 incorporating a series of site-directed spin labels (SDSL); these studies will be extended into the time domain using RFQ-EPR to probe larger scale motions of the spin-labeled active site loop in the same Co-L1(SDSL) series. This proposal will offer a new strategy for the characterization of reactions catalyzed by Zn(II)- containing enzymes. More specific to the M?Ls, our long term goal is to identify structural/mechanism details common to all M?Ls, and in the future, we plan to apply the approaches, developed in this proposal, to other distinct M?Ls. The methodologies developed herein will easily lend themselves to the study of other metalloenzymes, regardless of the identity, oxidation level, or spin state of the metal. PUBLIC HEALTH RELEVANCE: The emergence of antibiotic resistance in the clinic has resulted in a biomedical crisis in which bacterial infections, once treated with inexpensive antibiotics, are now untreatable and cause a large number of deaths annually in the US and throughout the world. This proposal describes experiments to probe a metallo-?-lactamase, which causes resistance to penicillin and other ?-lactam containing antibiotics in the clinic, as it proceeds during catalysis. The information gleaned in these studies can be used to guide in the future development of M?L inhibitors.

描述（由申请人提供）：本提案描述了在生物化学反应过程中探测酶结构变化的研究。我们提出利用速冻淬火（RFQ）与几种光谱技术相结合来探测金属-？内酰胺酶(M ?L)来自嗜麦芽寡养单胞菌的L1作为底物结合并转化为产物。具体地说,……在具体目标#1中，我们提出将快速冷冻猝灭（RFQ）技术与传统的光谱技术(包括x射线吸收光谱（XAS）、EPR和电子核双共振（ENDOR）相结合，探索L1与Zn和/或Co在活性位点与a ？内酰胺衬底。在具体目标#2中，我们将通过包含一系列位点导向自旋标签（SDSL）的L1共含衍生物的连续波EPR研究，研究翻转过程中覆盖金属位点的位置保守柔性环的溶液动力学；这些研究将使用RFQ-EPR扩展到时域，以探测相同Co-L1（SDSL）系列中自旋标记活性位点环的更大规模运动。这将为含锌酶催化反应的表征提供一种新的策略。具体到M？l，我们的长期目标是确定所有M？在未来，我们计划将本提案中开发的方法应用于其他不同的M？Ls。本文开发的方法将很容易地适用于其他金属酶的研究，而不考虑金属的特性、氧化水平或自旋状态。

项目成果

New β-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-β-lactamases.

• DOI: 10.1016/j.bmcl.2013.08.098
• 发表时间: 2013-11-01
• 期刊: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
• 影响因子: 2.7
• 作者: Yang, Ke-Wu;Feng, Lei;Yang, Shao-Kang;Aitha, Mahesh;LaCuran, Alecander E.;Oelschlaeger, Peter;Crowder, Michael W.
• 通讯作者: Crowder, Michael W.