Protein Engineering of PhUGT, a Donor Promiscuous Glycosyltransferase, for the Improved Enzymatic Synthesis of Antioxidant Quercetin 3-O-N-Acetylgalactosamine

Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymaticallysynthesized due to the lack of well-identifiedN-acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identifiedflavonoid 3-O-galactosyltransferase fromPetunia hybrida, was demonstrated to display quercetin GalNAc-T activity, transferring aN-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc witha low conversion of 11.7% at 40 degrees C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got anenhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidantactivity with other quercetin 3-O-glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase(GT), recognizing seven sugar donors. Thisfinding overturned a previous notion that PhUGT exclusively recognized UDP-galactose(UDP-Gal). The reason why PhUGT was mistaken for a UDP-Gal-specific GT was demonstrated to be a shorter reaction time, inwhich many quercetin 3-O-glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factoryexpressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundationfor the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.

Automated summary

In this study, Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a compound that had never been enzymatically synthesized before, was successfully produced. The enzyme PhUGT was identified to have quercetin GalNAc-T activity and was engineered to enhance the conversion rate. The study also revealed the donor promiscuity of PhUGT and provided a potent biocatalyst for glycodiversifying quercetin.