Characterization of a Novel Bifunctional UDP-Glycosyltransferase (PcUGT71BE11) from <i>Polygonum cuspidatum</i> in Emodin Glycosides Formation and Deglycosylation

Emodin glucosides, including emodins 1-<i>O</i>-glucoside (E1G), E6G, and E8G, possess significant pharmaceutical and nutraceutical potentials. Here, we identified PcUGT71BE11 from <i>Polygonum cuspidatum</i> as a bifunctional UGT capable of both glycosylating all three hydroxyl groups of emodin and deglycosylating E1G and E8G. Kinetic and time-course analyses showed that E1G was the first product, and its accumulation was pH-dependent. PcUGT71BE11 predominantly produced E1G at pH 8.8 but shifted to E6G at pH 6.8 due to decreased glycosylation and increased deglycosylation at acidic pH. The UDP-dependent deglycosylation was attenuated by sucrose synthase introduction, thereby enhancing E1G accumulation. Structural modeling, molecular dynamics, MM/GBSA binding free energy, and mutagenesis revealed the molecular basis: H19, D120, P190, and E395 governed the bifunctionality, while C144, I192, and L147 contributed to the regioselectivity. Notably, the L147N mutant enhanced the 8-<i>O</i>-glycosylation selectivity. This study elucidates the molecular mechanism of PcUGT71BE11 and provides a promising catalyst for producing diverse emodin glycosides.