Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.
Biotechnology Institute, St. Paul, Minnesota, USA.
Protein Sci. 2024 Apr;33(4):e4954. doi: 10.1002/pro.4954.
Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity 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 generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, 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. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa 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.
许多革兰氏阴性菌使用 N-酰基-L-高丝氨酸内酯 (AHL) 信号来协调表型，如生物膜形成和毒力因子产生。群体感应淬灭酶，如 AHL 酰基水解酶，化学降解这些分子，防止细菌接收信号并抑制不良的生物膜相关特征。这些特性使酰基水解酶成为控制微生物的有吸引力的候选物，但需要具有高活性水平和底物特异性且能够被制成材料的候选物。在这项工作中，我们使用 Protein One-Stop Shop Server 针对两种 AHL 酰基水解酶 PvdQ 和 MacQ 进行了工程改造，以产生这些改进的特性。酰基水解酶的工程改造受到低通量酶测定的限制。为了缓解这一挑战，我们报告了一种用于 AHL 酰基水解酶的实时动力学测定法，该测定法监测高丝氨酸内酯的实时产生。使用该测定法，我们鉴定了 PvdQ 的变体，这些变体显著稳定，熔点增加高达 13.2°C，这转化为对有机溶剂的高抗性和与材料涂层的更高兼容性。虽然 MacQ 突变体出乎意料地不稳定，但它们的动力学特性有了显著改善，对 N-丁酰基-L-高丝氨酸内酯和 N-己酰基-L-高丝氨酸内酯的增加超过 10 倍。因此，这些变化导致使用生物传感器模型的淬灭能力增加，并增加了对铜绿假单胞菌 PA14 毒力因子产生的抑制。虽然 MacQ 变体之一 M1 的晶体结构没有揭示明显的结构决定因素来解释观察到的动力学变化，但它允许捕获酰基-酶中间产物，该中间产物证实了 AHL 酰基水解酶先前假设的催化机制。
