Functional characterization,structural basis,and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis  

作　　者：Zilong Wang Xueqing Du Guo Ye Haotian Wang Yizhan Liu Chenrui Liu Fudong Li HansÅgren Yang Zhou Junhao Li Chao He De-An Guo Min Ye 

机构地区：[1]State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing 100191,China [2]Anhui Key Laboratory of Modern Biomanufacturing and School of Life Sciences,Anhui University,Hefei 230601,China [3]National Science Center for Physical Sciences at Microscale Division of Molecular&Cell Biophysics and School of Life Sciences,University of Science and Technology of China,Hefei 230026,China [4]Department of Physics and Astronomy,Uppsala University,Uppsala SE-75120,Sweden [5]School of Pharmacy,Jinan University,Guangzhou 510632,China [6]Shanghai Research Center for Modernization of Traditional Chinese Medicine,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China

出　　处：《Acta Pharmaceutica Sinica B》2024年第8期3746-3759,共14页药学学报（英文版）

基　　金：supported by the National Key Research and Development Program of China(No.2023YFA0914100);China National Postdoctoral Program for Innovation Talents(No.BX20220022);National Natural Science Foundation of China(No.82304326);Natural Science Foundation of Anhui Province(No.2008085MC92,China);the National Supercomputer Center(SNIC2022-3-34)at Linköping University(Sweden).

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(2.15Å)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 L/(s·μmol),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.

关 键 词：GLYCOSYLTRANSFERASE Regio-selectivity Crystal structure De-glycosylation Catalytic mechanisms 

分 类 号：R284.3[医药卫生—中药学]