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-glycosyl...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.展开更多
基金supported by the National Key Research and Development Program of China(No.2023YFA0914100)China National Postdoctoral Program for Innovation Talents(No.BX20220022)+2 种基金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).
文摘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.
