黄芪中三种黄酮类Glycosyltransferases的功能表征及AmUGT88E37的底物混杂机理分析

Flavonoid glycosylation is a key tailoring reaction involved in the accumulation and structural diversification of bioactive flavonoids in Astragalus membranaceusvar. mongholicus. In this study, three UDP-glycosyltransferases, AmUGT73AC19, AmUGT88E36, and AmUGT88E37, were identified and functionally characterized. Yeast-based assays and in vitro reactions showed that all three enzymes glucosylated calycosin and formononetin to produce calycosin-7-O-β-d-glucoside and ononin, respectively, in a metal ion-independent manner, with AmUGT88E36 and AmUGT88E37 showing higher conversion capacities. Substrate profiling revealed clear functional differentiation: AmUGT88E36 preferentially catalyzed isoflavone 7-O-glucosylation, AmUGT73AC19 displayed moderate regioselective flexibility, and AmUGT88E37 accepted isoflavones, flavonols, flavones, and dihydroflavones to generate 7-O-, 4'-O-, and 6-O-glucosides. Docking and mutagenesis identified Glu383 as critical for AmUGT88E37 activity and Val144, Tyr135, Leu195, and Val141 as contributors to regioselectivity. These findings elucidate the enzymatic basis of isoflavone glycoside formation in Astragalus membranaceusvar. mongholicus and provide promising biocatalytic tools for the regioselective synthesis of structurally diverse flavonoid glycosides.