New Insights into the Binding and Catalytic Mechanisms of <i>Bacillus thuringiensis</i> Lactonase: Insights into <i>B. thuringiensis</i> AiiA Mechanism

The lactonase enzyme (AiiA) produced by Bacillus thuringiensis serves to degrade autoinducer-1 (AI-1) signaling molecules in what is an evolved mechanism by which to compete with other bacteria. Bioassays have been previously performed to determine whether the AI-1 aliphatic tail lengths have any effect on AiiA’s bioactivity, however, data to date are conflicting. Additionally, specific residue contributions to the catalytic activity of AiiA provide for some interesting questions. For example, it has been proposed that Y194 serves to provide an oxyanion hole to AI-1 which is curious given the fact the substrate spans two Zn(2+) ions. These ions might conceivably provide enough charge to promote both ligand stability and the carbonyl activation necessary to drive a nucleophilic attack. To investigate these questions, multiple molecular dynamics simulations were performed across a family of seven acylated homoserine lactones (AHL) along with their associated intermediate and product states. Distance analyses and interaction energy analyses were performed to investigate current bioassay data. Our simulations are consistent with experimental studies showing that AiiA degrades AHLs in a tail length independent manner. However, the presence of the tail is required for activity. Also, the putative oxyanion hole function of Y194 toward the substrate is not observed in any of the reactant or product state simulation trajectories, but does seem to show efficacy in stabilizing the intermediate state. Last, we argue through ionization state analyses, that the proton shuttling necessary for catalytic activity might be mediated by both water and substrate-based intra-molecular proton transfer. Based on this argument, an alternate catalytic mechanism is proposed.苏云金芽孢杆菌（Bacillus thuringiensis）产生的内酯酶（lactonase, AiiA），其功能是降解自体诱导物-1（autoinducer-1, AI-1）信号分子，这是一种为与其他细菌竞争而演化出的机制。此前已有研究通过生物测定（bioassay）探究AI-1的脂肪族侧链长度是否会对AiiA的生物活性产生影响，但截至目前相关实验数据仍存在矛盾。此外，AiiA催化活性相关的特定残基的作用机制也引发了诸多值得探讨的问题。例如，有研究提出Y194可向AI-1提供氧负离子孔（oxyanion hole），但考虑到底物跨越两个二价锌离子（Zn²⁺），这一推论颇为令人费解：理论上这两个离子本可提供足够的电荷，以促进配体稳定并驱动亲核攻击所需的羰基活化。为探究上述问题，本研究针对7种酰化高丝氨酸内酯（acylated homoserine lactones, AHL）家族及其对应的中间态与产物态，开展了多组分子动力学模拟（molecular dynamics simulation）。研究通过距离分析与相互作用能量分析，对当前已有的生物测定数据进行了验证与分析。本研究的模拟结果与已有实验研究相符：AiiA降解AHL的过程不受侧链长度影响，但侧链的存在是其发挥催化活性的必要前提。此外，在所有反应物或产物态的模拟轨迹中，均未观察到Y194对底物发挥推定的氧负离子孔功能，但该残基似乎可有效稳定反应中间态。最后，通过电离状态分析，本研究提出催化活性所需的质子穿梭（proton shuttling）可能由水分子与底物介导的分子内质子转移共同完成，并基于此提出了一种替代的催化机制。