Zhao Jingrong, Xu Youqiang, Lu Hongyun, Zhao Dong, Zheng Jia, Lin Mengwei, Liang Xin, Ding Ze, Dong Wenqi, Yang Maochen, Li Weiwei, Zhang Chengnan, Sun Baoguo, Li Xiuting
Key Laboratory of Brewing Microbiome and Enzymatic Molecular Engineering, China General Chamber of Commerce, Beijing Technology and Business University, Beijing, China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing, China.
Front Microbiol. 2023 Jan 12;13:1107104. doi: 10.3389/fmicb.2022.1107104. eCollection 2022.
Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu. YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to α/β fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The π-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu.
脂肪酸乙酯是浓香型白酒中重要的风味物质。YJX - 8被认为是发酵过程中酯合成的重要微生物。来自YJX - 8的LIP05酶能在水相中高效催化脂肪酸乙酯的合成，但影响酯化反应的关键催化位点尚不清楚。本研究结合同源建模、分子动力学模拟、分子对接和定点突变来分析LIP05的催化机制。蛋白质结构建模表明LIP05属于α/β折叠水解酶，包含一个盖子结构域和一个具有保守催化三联体Ser150 - His215 - Asp202的核心催化口袋，以及由Gly73和Thr74组成的氧阴离子洞。发现盖子结构域的Ile30和Leu37影响底物特异性。Tyr116和Tyr149之间的π键堆积在稳定LIP05的催化活性中心方面起重要作用。Tyr116和Ile204通过构成底物进入通道决定底物谱。Leu83、Ile204、Ile211和Leu216残基通过空间位阻和疏水相互作用参与形成疏水底物结合口袋。提出了LIP05在水相中酯化反应的催化机制，为阐明浓香型白酒发酵过程中脂肪酸乙酯的合成提供了参考。
