Functional Characterization of PcUGT73BF6 from <i>Polygonum Cuspidatum</i> and the Facilitation of Emodin Catalysis via Site-Directed Mutagenesis

Emodin (Emo) glycosides are significant bioactive constituents of <i>Polygonum cuspidatum</i>, a well-known dietary plant in China and Japan. However, the role of uridine diphosphate glycosyltransferases (UGTs) in Emo glycoside biosynthesis in <i>P. cuspidatum</i> remains elusive, which greatly hampers their industrial production. Here, first, we successfully identified an Emo glycosyltransferase, PcUGT73BF6, capable of regioselectively forming Emo 6-<i>O</i>-glucoside, and exhibited sugar donor promiscuity with a preference for UDP-Glc. Then, G148 in a flexible loop correlated with the donor selectivity in PcUGT73BF6 was revealed as the key contributor to the abovementioned donor selectivity and preference. Besides, we found that the mutant S394G showed a novel enzymatic characteristic of glycosylating multiple hydroxyl groups of Emo, and the spacious binding pocket of S194G/S394G was the main underlying basis of enhanced catalytic efficiency. Our study revealed the molecular mechanism of PcUGT73BF6 for donor selectivity and also provided promising catalysts for the industrial production of structurally diverse Emo <i>O</i>-glycosides.