期刊
ACS CATALYSIS
卷 11, 期 24, 页码 14781-14790出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.1c04191
关键词
directed evolution; glycosyltransferase; regioselectivity; iterative saturation mutagenesis; flavonoids
资金
- National Natural Science Foundation of China [21775040, 22077029, 22034002]
- Science Fund for Distinguished Young Scholars of Hunan Province [2021JJ10034]
- Huxiang High-Level Talent Gathering Project of Hunan Province [2019RS1040]
- Natural Science Foundation of China [31800638]
- high-performance public computing service platform of Jinan University
Plant glycosyltransferase MiCGT was engineered by directed evolution to catalyze the glycosylation of flavonoids for pharmaceutical applications, resulting in mutants with enhanced selectivity and activity towards seven different flavonoids. The best quadruple mutant VFAH showed strict 3-O glycosylation selectivity and a 120-fold activity enhancement towards the model substrate quercetin.
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 chemoand 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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