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

Abstract

Plant bioactive metabolites such as flavonoids are usually present in glycosylated forms by the attachment of various sugar groups. In this study, a catalytically flexible and reversible glycosyltransferase (HtUGT72AS1) was cloned and characterized from Helleborus thibetanus. HtUGT72AS1 could directly accept six sugar donors (UDP-glucose/-arabinose/-galactose/-xylose/-Nacetylglucosamine/-rhamnose) to catalyze the 3-OH glycosylation of flavonols. It also catalyzed the 4′ and 7-OH glycosylation of other types of flavonoids, which lacked the 3-OH group. Additionally, the HtUGT72AS1-catalyzed reaction was highly reversible when using 2-chloro-4-nitrophenyl glycosides as substrates, which could be used for one-pot or coupled production of bioactive glycosides. It is the first reported UGT for the synthesis of arabinosides and galactosides using a transglycosylation platform. Based on structural modeling and mutagenetic analysis, the mutation of Tyr377 to Ara enhanced the catalytic efficiency of HtUGT72AS1 toward UDP-N-acetylglucosamine, and the V146S mutant gained an improvement in the regioselectivity toward 7-OH of flavonoids.

The sugar donor specificity of plant family 1 glycosyltransferases

“…UGTs that can accept several UDP-sugars serve as the epitome in glycodiversification ( Wen et al, 2023 ; Wu et al, 2023 ). Hence, it is important to understand which traits lead to UDP-sugar promiscuity.…”

“…Gly15Ser, Gly15Phe, and Gly15Val all significantly decreased UDP-Gal and UDP-GlcNAc activity which supported the hypothesis of the cavity volume playing a significant role in the donor promiscuity of Sb 3GT1 ( Figure 3D ). Similarly, Wen et al tried to alter the donor substrate scope of the promiscuous UGT72AS1 from Helleborus thibetanus through site-directed mutagenesis by comparing the sequence information with sequences of both donor promiscuous and donor-specific UGTs ( Wen et al, 2023 ). However, constructed mutants either exhibited conservation of function with all tested donors or a significant decrease with all donor substrates.…”