Chemical tools to investigate chain-flipping in quorum signal synthases

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调节关键生理功能 包括毒力、生物膜形成和毒素产生在内的活动。AHL合酶使AHL 酶偶联酰基-酸性磷酸酶和S-腺苷-L-蛋氨酸代谢物的自动诱导信号 促进细菌的群体感应。因此，AHL合成酶抑制剂具有重要的应用前景 用于治疗多重耐药细菌感染的抗菌药。设计AHL合成酶特异性抑制剂， 然而，这仍然是一个巨大的挑战。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，使其有别于其他利用酰基-ACP或 萨姆基板。合成酶的一个独特方面是它能够选择性地结合和反应一种 特定的酰基-ACP底物，从而增强了AHL信号合成的保真度。一般而言，酰基-ACP结合 到配对酶是一个最小的两步过程，包括ACP酸的初始静电对接 残基在合成酶的碱性补丁上(蛋白质-蛋白质结合)，然后是酰基- 从ACP到酶活性部位口袋的链状货物通过一种被称为“链式翻转”的过程。 到目前为止，由于缺乏将绑定与链翻转步骤分离的方法，因此不可能实现 能够独立报告ACP中利用酶反应的链翻转步骤。要解决这个问题 限制，我们在Esai AHL合成酶上放置了位点特异的荧光标记，进行了酶-ACP 用停流荧光计滴定稳态前动力学区域并测定Kon和Koff 用于荧光和非荧光货物链从ACP翻转到ESAI合成酶。建立在 我们的初步数据，我们在本申请中提出了两个目标来验证基于荧光的方法 AHL合成酶中翻链速率常数的测定。圆满完成拟议中的 研究应加强我们对AHL底物识别的机制基础的理解 合酶b)有助于建立筛选AHL合酶抑制剂的基于荧光的HTS方法 以及c)为AHL合成中链翻转步骤的单分子酶学研究打开大门 以及d)促进这一工具的部署，以研究更广泛范围内的链式翻转动力学 医学上重要的、依赖于载体蛋白的酶。

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.