Mechanism of the Humicola insolens Cel7B E197S mutant catalyzed flavonoid glycosides synthesis: a QM/MM metadynamics simulation study
Mechanism of the Humicola insolens Cel7B E197S mutant catalyzed flavonoid glycosides synthesis: a QM/MM metadynamics simulation study
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DOI：
10.1007/s00214-013-1367-3
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发表时间：
2013-04
影响因子：
1.7
通讯作者：
Yu Zhang;Shihai Yan;L. Yao
中科院分区：
文献类型：
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作者：
Yu Zhang;Shihai Yan;L. Yao
用QM/MM元动力学模拟方法研究了腐霉菌Cel7B E197S催化木犀草素与α-乳糖基氟化物糖基化反应的反应路径。催化是通过顺序机制进行的。首先，质子从糖基受体木犀草素转移到催化酸碱残基E202。其次，糖基供体α-乳糖基氟化物的C1-F共价键在S197的羟基的帮助下被切割，F-离子捕获了D199的羧基质子，而−1亚基上的糖环的构象从椅子变成了半椅子。第三，木犀草素阴离子的O‘与乳糖基的C_1形成了新的糖苷键，形成了−-1糖基环的1，4B船型构象。限速步骤是C_1-F键断裂，势垒约为25.9千卡/摩尔。分析了反应中活性中心的几何参数和氢键的变化，揭示了残基S197、D199和E202的重要作用。
The reaction path ofHumicola insolensCel7B E197S catalyzed glycosylation of luteolin by α-lactosyl fluoride was investigated using QM/MM metadynamics simulations. The catalysis proceeds via a sequential mechanism. Firstly, a proton transfers from the glycosyl acceptor, luteolin, to the catalytic acid/base residue, E202. Secondly, the C1–F covalent bond of the glycosyl donor, α-lactosyl fluoride, is cleaved with the assistance of the hydroxyl group of S197, and the F anion captures the carboxylic proton of D199, while the conformation of the glycosyl ring at −1 subsite changes from a chair to a half-chair. Thirdly, a new glycosidic bond is formed between C1of the lactosyl cation and O4′ of the luteolin anion during which a1,4B boat conformation comes into being for the subsite −1 glycosyl ring. The rate-limiting step is the C1–F bond cleavage with a barrier of ~25.9 kcal/mol. The change of geometric parameters and hydrogen bonds in the active site along the reaction is analyzed, revealing the important role of residues S197, D199, and E202.