Sompiyachoke Kitty, Elias Mikael H
University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, St. Paul, MN, 55108, USA.
University of Minnesota, Biotechnology Institute, St. Paul, MN, 55108, USA.
bioRxiv. 2023 Sep 1:2023.09.01.555929. doi: 10.1101/2023.09.01.555929.
Many Gram-negative bacteria respond to acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as acylases, chemically degrade AHL signals, prevent signal reception by bacteria, and inhibit undesirable traits related to biofilm. These capabilities make these enzymes appealing candidates for controlling microbes. Yet, enzyme candidates with high activity levels, high substrate specificity for specific interference, and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to obtain improved acylase variants. The engineering of acylase is complicated by low-throughput enzymatic assays. To alleviate this challenge, we report a time-course kinetic assay for AHL acylase that tracks the real-time production of homoserine lactone. Using the protein one-stop shop server (PROSS), we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2 °C, which translated into high resistance against organic solvents and increased compatibility with material coatings. We also generated mutants of MacQ with considerably improved kinetic properties, with >10-fold increases against -butyryl-L-homoserine lactone and hexanoyl-L-homoserine lactone. In fact, the variants presented here exhibit unique combinations of stability and activity levels. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.
许多革兰氏阴性菌会对酰基-L-高丝氨酸内酯（AHL）信号作出反应，以协调生物膜形成和毒力因子产生等表型。群体感应淬灭酶，如酰基酶，能化学降解AHL信号，阻止细菌接收信号，并抑制与生物膜相关的不良特性。这些特性使这些酶成为控制微生物的有吸引力的候选者。然而，需要具有高活性水平、对特定干扰具有高底物特异性且能够配制成材料的酶候选物。在这项工作中，我们对两种AHL酰基酶PvdQ和MacQ进行了工程改造，以获得改良的酰基酶变体。酰基酶的工程改造因低通量酶促测定而变得复杂。为了应对这一挑战，我们报告了一种用于AHL酰基酶的时间进程动力学测定法，该方法可追踪高丝氨酸内酯的实时产生。使用蛋白质一站式服务器（PROSS），我们鉴定出了PvdQ的变体，其稳定性显著提高，熔点升高高达13.2℃，这转化为对有机溶剂的高抗性以及与材料涂层的相容性增加。我们还生成了MacQ的突变体，其动力学特性有了显著改善，对丁酰-L-高丝氨酸内酯和己酰-L-高丝氨酸内酯的活性提高了10倍以上。事实上，这里展示的变体表现出稳定性和活性水平的独特组合。因此，这些变化导致使用生物传感器模型时淬灭能力增强，对PA14毒力因子产生的抑制作用更大。虽然MacQ变体之一M1的晶体结构没有揭示出明显的结构决定因素来解释观察到的动力学变化，但它捕获了一种酰基酶中间体，证实了先前假设的AHL酰基酶的催化机制。
