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.

摘要 革兰氏阴性菌利用酰基-高丝氨酸内酯介导的群体感应来调节关键的生理 包括毒力、生物膜形成和毒素产生的活性。阿勒酶使阿勒 通过酶促偶联酰基-ACP和S-腺苷-L-甲硫氨酸代谢物， 促进细菌的群体感应。因此，阿勒合酶抑制剂具有重要的前景， 抗生素治疗多重耐药细菌感染。设计阿勒合酶特异性抑制剂， 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 阿勒脱氢酶的功能方面，其将它们与利用酰基-ACP或 SAM底物。合酶的一个独特方面是它能够选择性地结合并与 特异性酰基-ACP底物，从而增强阿勒信号合成的保真度。一般来说，酰基-ACP结合 是一个最小的两步过程，涉及ACP酸性的初始静电对接， 残基转移到合酶的碱性补丁上（蛋白质-蛋白质结合），然后酰基- 链货物从ACP到酶的活性位点口袋通过称为“链翻转”的过程。 直到现在，由于缺乏方法， 能够独立报道利用酶反应的ACP中的链翻转步骤。为了解决这个 限制，我们放置了一个位点特异性荧光标记的EsaI阿勒合酶，进行酶-ACP 使用停流荧光计在预稳态动力学方案中进行滴定 对于从ACP到EsaI合酶的荧光和非荧光货物链翻转。基础上 根据我们的初步数据，我们在本申请中提出了两个目的，以验证基于荧光的方法， 测定阿勒合酶中的链翻转速率常数。圆满完成拟议的 研究应该：a）加强我们对阿勒中底物识别的机制基础的理解 酶B）帮助开发用于筛选阿勒合酶抑制剂的基于荧光的HTS测定 以及c）为阿勒合成中链翻转步骤的单分子酶学研究打开大门 和d）促进该工具的部署，以在更宽的范围内研究链翻转动力学， 医学上重要的载体蛋白依赖酶。