College of Food and Pharmaceutical Sciences , Ningbo University , Ningbo 315211 , P. R. China.
Key Laboratory of Animal Protein Food Deep Processing Technology of Zhejiang Province , Ningbo University , Ningbo 315211 , P. R. China.
J Agric Food Chem. 2020 Feb 26;68(8):2516-2527. doi: 10.1021/acs.jafc.9b07833. Epub 2020 Feb 17.
In the Gram-negative bacterium , -acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the gene from the isolate HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces -butanoyl-l-homoserine lactone (C-HSL) and -hexanoyl-l-homoserine lactone (C-HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C-HSL using -adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a β bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C-ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.
在革兰氏阴性菌中，-酰基高丝氨酸内酯（AHL）介导的群体感应（QS）影响致病性、蛋白质分泌和运动性。然而，AHL 生物合成的催化机制以及 AhyI 成员的结构基础和底物特异性仍不清楚。在本研究中，我们从分离株 HX-3 中克隆了基因，通过超高效液相色谱-串联质谱（UPLC-MS/MS）分析证实，过表达的 AhyI 蛋白可产生六种类型的 AHL，与之前报道的 AhyI 仅产生 -丁酰基-l-高丝氨酸内酯（C-HSL）和 -己酰基-l-高丝氨酸内酯（C-HSL）不同。体外生物合成测定的结果表明，纯化的 AhyI 可以使用 -腺苷甲硫氨酸（SAM）和丁酰基酰基载体蛋白（ACP）作为底物催化 C-HSL 的形成，并表明 AhyI 介导的 AHL 合成中使用的脂肪酰基底物源自酰基-ACP，而不是酰基辅酶 A。AhyI 以丁酰基-ACP 作为酰基底物的动力学数据表明，HX-3 AhyI 酶的催化效率与其他 LuxI 同系物相当。在本研究中，首次对 AhyI 的三级结构建模结果以及分子对接和结构功能分析结果表明，几个关键残基以及与酰化相关的二级结构的重要性。一个 Phe125-Phe152 夹抓住末端甲基以协助稳定假定结合口袋中的长酰基链。在强疏水环境中的堆积相互作用、氢键网络和β 凸块可能稳定 ACP 酰基链，使 SAM α-胺攻击硫酯碳，为 AhyI 如何合成具有不同酰链长度的 AHL 提供了相对合理的解释。此外，Trp34 参与形成 C-ACP 的结合口袋，并在 SAM 结合时变得有序，为催化提供了良好的基础。AhyI 既能产生短链 AHL 又能产生长链 AHL 的新发现增强了当前对该酶产生的各种 AHL 的认识。这些结构数据有望为 AhyI 的 AHL 合成提供分子基础。
