区域选择性
糖基化
糖基转移酶
饱和突变
定向进化
化学
蛋白质工程
突变体
化学选择性
生物化学
催化作用
基质(水族馆)
组合化学
立体化学
生物
酶
基因
生态学
作者
Zexing Wen,Zhimin Zhang,Liang Zhong,Jiaqian Fan,Min Li,Yuanhong Ma,Yang Zhou,Wei Zhang,Bin Guo,Bo Chen,Jian‐bo Wang
出处
期刊:ACS Catalysis
日期:2021-11-25
卷期号:11 (24): 14781-14790
被引量:36
标识
DOI:10.1021/acscatal.1c04191
摘要
Glycosyltransferases have attracted increasing interest for the ability to construct glycosylated molecules in a facile way. However, promiscuous chemoselectivity and poor regioselectivity hinder their widespread application in the synthetic field, especially in the pharmaceutical area. Here, a plant glycosyltransferase, MiCGT, was engineered by directed evolution to catalyze the glycosylation of flavonoids, which opens the door to pharmaceutical applications. Combining an alanine scan and iterative saturation mutagenesis, mutants with enhanced chemo- and regioselectivity and significantly improved activities toward seven different flavonoids were evolved, and two glycosylated products were prepared on a large scale. The best quadruple mutant VFAH enables strict 3-O glycosylation selectivity and a 120-fold activity enhancement toward the model substrate quercetin relative to the wild type (WT). Moreover, the crystal structures of the WT and mutant VFAH were obtained, a breakthrough of its kind in plant glycosyltransferase research. The origin of substrate specificity and regioselectivity was elucidated by combining the experimental data with the unique structure information. We anticipate that this work will aid future protein engineering of this type of enzyme.
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