Functional Divergence of Three Segmentally Duplicated UGT71 Enzymes Reveals Distinct Glycosylation Strategies toward Resveratrol and Emodin in Polygonum cuspidatum

Polygonum cuspidatum is a medicinal and edible plant rich in bioactive phenolics, predominantly occurring as O -glycosides catalyzed by UDP-glycosyltransferases (UGTs). However, how gene duplication drives the UGT functional diversification remains unclear. Here, 186 PcUGTs were identified and classified into 17 phylogenetic groups with segmental duplication as the primary driver of family expansion. Two segmentally duplicated paralogs of the previously characterized PcUGT71BE11, namely, PcUGT71AH6 and PcUGT71U25, exhibited pronounced functional divergence. PcUGT71AH6 showed no detectable glycosylation activity toward resveratrol or emodin under the tested conditions. In contrast, PcUGT71U25 displayed site-promiscuous glycosylation toward emodin and UDP-dependent deglycosylation activity toward its glycosides. Docking and molecular dynamics simulations revealed a conformationally adaptable active pocket in PcUGT71U25 that accommodates multiple productive emodin binding poses. Notably, the E86A mutation enhanced C1-OH site selectivity by reshaping substrate binding conformations. Overall, this study provides mechanistic insights into UGT diversification and phenolic glycoside biosynthesis in P. cuspidatum .