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

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外文摘要：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 A)was solved,and mechanisms of its regio-selectivity was interpreted by pAa 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·pmol),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 bio-catalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

外文关键词：

GlycosyltransferaseRegio-selectivityCrystal structureDe-glycosylationCatalytic mechanisms

作者：

Zilong Wang、Xueqing Du、Guo Ye、Haotian Wang、Yizhan Liu、Chenrui Liu、Fudong Li、Hans ?gren、Yang Zhou、Junhao Li、Chao He、De-An Guo、Min Ye

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作者单位：

State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing 100191,China

Anhui Key Laboratory of Modern Biomanufacturing and School of Life Sciences,Anhui University Hefei 230601,China

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

Department of Physics and Astronomy,Uppsala University,Uppsala SE-751 20,Sweden

School of Pharmacy,Jinan University,Guangzhou 510632,China

Shanghai Research Center for Modernization of Traditional Chinese Medicine,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China

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基金：

National Key Research and Development Program of ChinaChina National Postdoctoral Program for Innovation TalentsNational Natural Science Foundation of ChinaNatural Science Foundation of Anhui Province,ChinaSwedish National Infrastructure for Computing(SNIC)at the National Supercomputer Center at Link?ping University(Sweden)

项目编号：

2023YFA0914100BX20220022823043262008085MC92SNIC2022-3-34

出版年：

2024

DOI：

10.1016/j.apsb.2024.04.001

药学学报(英文版)

CSTPCD

ISSN：

年,卷(期)：2024.14(8)