Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition  

作　　者：Jieren Liao Guangxin Sun Elisabeth Kurze Wieland Steinchen Timothy D.Hoffmann Chuankui Song Zhiwei Zou Thomas Hoffmann Wilfried G.Schwab 

机构地区：[1]Biotechnology of Natural Products,Technische University at Müunchen,Liesel-Beckmann-Str.1,85354 Freising,Germany [2]Center for Synthetic Microbiology(SYNMIKRO)&Faculty of Chemistry,Philipps-University Marburg,Karl-von-Frisch-Straße 14,35043 Marburg,Germany [3]State Key Laboratory of Tea Plant Biology and Utilization,International Joint Laboratory on Tea Chemistry and Health Effects,Anhui Agricultural University,230036 Hefei,Anhui,P.R.China [4]Present address:Department of Molecular Biophysics and Biochemistry,Yale University,New Haven,CT,USA

出　　处：《Plant Communications》2023年第3期151-170,共20页植物通讯（英文）

基　　金：DFG SCHW 634/34-1.

摘　　要：Uridine diphosphate-dependent glycosyltransferases(UGTs)mediate the glycosylation of plant metabolites,thereby altering their physicochemical properties and bioactivities.Plants possess numerous UGT genes,with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics,but the biological function and molecular basis of these phenomena are not fully understood.The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition(Ki)at 4 mM scopoletin,whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 mM.We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs.A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reducedscopoletin substrate inhibition,whereas its monolignolgly cosylation activity was almost unaffected.Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation,leading to enhanced promiscuity,which was accompanied by substrate inhibition.Studies of 3D structures identified open and closed UGT conformers,allowing visualization of the dynamics of conformational changes that occur during catalysis.Previously postulated substrate access tunnels likely serve as drainage channels.The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release.Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions.The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

关 键 词：GLYCOSYLTRANSFERASE substrate inhibition hydrogen/deuterium exchange mass spectrometry protein morphing protein conformer SCOPOLETIN 

分 类 号：Q94[生物学—植物学]