Functional characterization,structural basis,and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis
作者机构：State Key Laboratory of Natural and Biomimetic DrugsSchool of Pharmaceutical SciencesPeking UniversityBeijing 100191China Anhui Key Laboratory of Modern Biomanufacturing and School of Life SciencesAnhui UniversityHefei 230601China National Science Center for Physical Sciences at Microscale Division of Molecular&Cell Biophysics and School of Life SciencesUniversity of Science and Technology of ChinaHefei 230026China Department of Physics and AstronomyUppsala UniversityUppsala SE-75120Sweden School of PharmacyJinan UniversityGuangzhou 510632China Shanghai Research Center for Modernization of Traditional Chinese MedicineShanghai Institute of Materia MedicaChinese Academy of SciencesShanghai 201203China
出 版 物：《Acta Pharmaceutica Sinica B》 (药学学报（英文版）)
年 卷 期：2024年第14卷第8期
页 面：3746-3759页
核心收录：
学科分类：1008[医学-中药学(可授医学、理学学位)] 1006[医学-中西医结合] 100602[医学-中西医结合临床] 10[医学]
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)
主 题：Glycosyltransferase Regio-selectivity Crystal structure De-glycosylation Catalytic mechanisms
摘 要：Glycosylation is an important post-modification reaction in plant secondary metabolism,and contributes to structural diversity of bioactive natural *** plants,glycosylation is usually catalyzed by *** 2′-O-glycosides are rare ***,no UGTs have been reported,thus far,to specifically catalyze 2′-O-glycosylation of *** this work,UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-*** could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new *** of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase *** 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 *** 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 *** 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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