糖基化
化学
糖基转移酶
糖基
合理设计
类黄酮
立体化学
酶动力学
生物化学
糖苷
酶
活动站点
生物
抗氧化剂
遗传学
作者
Zilong Wang,Xueqing Du,Yanbin Guo,Haotian Wang,Y.F Liu,Chen-Rui Liu,Fudong Li,Hans Ågren,Yang Zhou,Junhao Li,Chao He,De‐An Guo,Min Ye
标识
DOI:10.1016/j.apsb.2024.04.001
摘要
Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2′-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2′-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pKa calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2′-O-glycosylation (kcat/Km, 0.23 μmol/(min·mg), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.
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