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

Abstract

Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymatically synthesized due to the lack of well-identified N-acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identified flavonoid 3-O-galactosyltransferase from Petunia hybrida, was demonstrated to display quercetin GalNAc-T activity, transferring a N-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc with a low conversion of 11.7% at 40 °C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got an enhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidant activity with other quercetin 3-O-glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase (GT), recognizing seven sugar donors. This finding 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, in which many quercetin 3-O-glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factory expressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundation for the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.

Glycosylation of Aromatic Glycosides by a Promiscuous Glycosyltransferase UGT71BD1 from Cistanche tubulosa

“…Uridine diphosphate (UDP) glycosyltransferase (UGT) is the key enzyme responsible for natural product glycosylation which transfers glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones). In the past decades, many UGTs have been identified from the model plants, , agricultural plants, − as well as various medicinal plants. − Among them, there are no lack of examples that possess notable catalysis promiscuity toward diverse kinds of natural substrates. − However, the substrate spectrum of most of the currently reported promiscuous UGTs originating from plants mainly focused on natural aglycones, such as phenylpropanoids, terpenoids, steroids, and flavonoids . UGTs that could accept diverse glycosides as substrates to catalyze further glycosylation reactions are rarely reported.…”

Exploring the Catalytic Flexibility and Reversibility of Plant Glycosyltransferase HtUGT72AS1 for Glycodiversification of Phenolic Compounds

“…Overall, HtUGT72AS1 has a higher capacity to utilize UDP-Glc/-Ara/-Xyl than other donors. In contrast, flavonoid UGTs are commonly selective for one type of UDP-sugar or glycosylate at a specific hydroxyl position. ,, The catalytic flexibility of HtUGT72AS1 makes it suitable for the glycodiversification of flavonoids and other phenolic compounds.…”

Novel Sesquiterpene and Diterpene Aminoglycosides from the Deep-Sea-Sediment Fungus Trichoderma sp. SCSIOW21

“…The reactions observed in the post tailoring steps may include complex oxidation, dehydration, and glycosylation ( Figure 4 ). Notably, the N-acetylaminosugar usually needs to undergo additional transamination and acetylation reactions prior to the glycosylation reaction [ 31 , 32 ].…”