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 Schwab · 发表于：Plant Communications · 年份：2022 · DOI：10.1016/j.xplc.2022.100506 · 被引用次数：15 · 研究领域：Plant Gene Expression Analysis、Plant biochemistry and biosynthesis、Toxin Mechanisms and Immunotoxins

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 μM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 μM. 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 reduced scopoletin substrate inhibition, whereas its monolignol glycosylation 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 secon...