Chemical tools to investigate chain-flipping in quorum signal synthases

研究群体信号合酶链翻转的化学工具

• 批准号: 10645548
• 负责人: Rajesh Nagarajan
• 金额: $ 21.75万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-23 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10645548
• 关键词: Active Sites; Acyl Carrier Protein; Address; Affinity; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biological Assay; Biological Models; Bromides; Carrier Proteins; Cells; Chemicals; Core Protein; Coupling; Cysteine; Data; Development; Docking; Electrostatics; Enzymes; Fluorescence; Genetic Code; Gram-Negative Bacteria; Investigation; Kinetics; Label; Laboratories; Learning; Literature; Measures; Mediating; Medicine; Methods; Microbial Biofilms; Molecular; Multi-Drug Resistance; Mutation; Physiological; Positioning Attribute; Preparation; Process; Production; Proteins; Reaction; Reporting; Research; S-Adenosylhomocysteine; S-Adenosylmethionine; Signal Transduction; Site; Specificity; Sulfhydryl Compounds; Titrations; Toxin; Tryptophan; Virulence; antimicrobial; crosslink; design; fluorophore; high throughput screening; homoserine lactone; in vivo; inhibitor; mutant; novel; prevent; quorum sensing; screening; single molecule; small molecule; small molecule inhibitor; tool

Abstract Gram-negative bacteria use acyl-homoserine lactone mediated quorum sensing to regulate key physiological activities that includes virulence, biofilm formation and toxin production. AHL synthases make AHL autoinducer signals by enzymatically coupling acyl-ACP and S-adenosyl-L-methionine metabolites to facilitate quorum sensing for the bacterium. Therefore, AHL synthase inhibitors hold significant promise as antimicrobials in treating multidrug resistant bacterial infections. Designing AHL synthase specific inhibitors, however, does remain a significant challenge. To develop QS selective inhibitors, we investigate unique functional aspects of AHL synthases that distinguish them from other enzymes utilizing either acyl-ACP or SAM substrates. One such unique aspect of the synthase is it’s ability to selectively bind and react with a specific acyl-ACP substrate thereby enforcing fidelity in AHL signal synthesis. In general, acyl-ACP binding to a partner enzyme is a minimal two-step process involving an initial electrostatic docking of ACP acidic residues on to a basic patch in the synthase (protein-protein binding) followed by the translocation of the acyl- chain cargo from the ACP to the enzyme’s active site pocket through a process referred to as “chain-flipping”. Decoupling the binding from the chain-flipping step was not possible until now due to lack of methods that could independently report on the chain flipping step in ACP utilizing enzyme reactions. To address this limitation, we placed a site-specific fluorescent label on the EsaI AHL synthase, conducted enzyme-ACP titrations in the pre-steady state kinetics regime using a stopped flow fluorometer and determined kon and koff for both fluorescent and non-fluorescent cargo chain flipping from ACP to the EsaI synthase. Building upon our preliminary data, we propose two aims in this application to validate a fluorescence based method for determining chain-flipping rate constants in AHL synthase enzymes. Successful completion of the proposed studies should a) enhance our understanding on the mechanistic basis of substrate recognition in AHL synthases b) aid in development of a fluorescence-based HTS assay for screening AHL synthase inhibitors and c) open doors for single molecule enzymological investigations on chain-flipping step in AHL synthesis and d) facilitate deployment of this tool to investigate chain-flipping kinetics over a broader range of medicinally important, carrier protein dependent enzymes.

抽象的 革兰氏阴性细菌利用酰基高丝氨酸内酯介导的群体感应来调节关键的生理功能 活动包括毒力、生物膜形成和毒素产生。 AHL 合成酶制造 AHL 通过酶促耦合酰基-ACP 和 S-腺苷-L-甲硫氨酸代谢物来产生自诱导信号 促进细菌的群体感应。因此，AHL 合酶抑制剂具有重要的前景： 抗菌药物治疗多重耐药细菌感染。设计 AHL 合酶特异性抑制剂， 然而，这仍然是一个重大挑战。为了开发 QS 选择性抑制剂，我们研究了独特的 AHL 合酶的功能方面将其与其他利用酰基-ACP 或酰基-ACP 的酶区分开来 SAM 基板。合酶的一个独特方面是它能够选择性地与 特异性酰基-ACP 底物，从而增强 AHL 信号合成的保真度。一般来说，酰基-ACP 结合 与伴侣酶的结合是一个最小的两步过程，涉及 ACP 酸性的初始静电对接 残基到合酶中的基本补丁上（蛋白质-蛋白质结合），然后酰基易位 通过称为“链翻转”的过程将货物从 ACP 链到酶的活性位点口袋。 由于缺乏方法，将链翻转步骤中的结合解耦直到现在才成为可能。 可以利用酶反应独立报告 ACP 中的链翻转步骤。为了解决这个问题 限制，我们在EsaI AHL合酶上放置了位点特异性荧光标记，进行酶-ACP 使用停流荧光计在预稳态动力学状态下进行滴定并确定 kon 和 koff 用于从 ACP 到 EsaI 合酶的荧光和非荧光货物链翻转。建立在 根据我们的初步数据，我们在此应用中提出了两个目标，以验证基于荧光的方法 确定 AHL 合酶中的链翻转速率常数。顺利完成拟议的 研究应该 a) 增强我们对 AHL 底物识别机制基础的理解 合酶 b) 帮助开发基于荧光的 HTS 测定法，用于筛选 AHL 合酶抑制剂 c) 为 AHL 合成中链翻转步骤的单分子酶学研究打开大门 d) 促进该工具的部署，以研究更广泛范围内的链翻转动力学 具有药用价值的载体蛋白依赖性酶。