Broaden the sugar donor selectivity of blackberry glycosyltransferase UGT78H2 through residual substitutions

Glycosylated secondary metabolites constitute a large proportion of nutrients or ingredients in consumed plants and related products. The glycosyl decoration largely depends on the activity of plant UDP-glycosyltransferases (UGTs). Mechanisms underlying the substrate selectivity and specificity of these reactions remain elusive. Here we report the cloning and functional characterization of a UGT, UGT78H2 in blackberry fruits. In vitro enzyme substrate specificity analysis and enzymatic kinetics evidenced that UGT78H2 glycosylate exclusively quercetin using uridine-5' diphosphate glucuronic acid (UDP-glucuronic acid) and uridine-5' diphosphate galactose (UDP-galactose). Site-directed mutagenesis was introduced into two residuals (N340P, K360N) previously unexplored. The mutation enhanced the protein catalyzing efficiency, especially toward UDP-galactose (23% higher), and expanded the sugar donor selectivity, which can use UDP-glucose as well. Molecular modeling and biochemical analysis results enable identification of the 23rd residue (360th in UGT78H2) of the PSPG (plant secondary product glycosyltransferase) motif as a key residue in defining this sugar selecting spectrum. Additionally, promoter of UGT78H2 was obtained. Transgenic analysis using the UGT78H2pro::GUS reporter system demonstrated that transcripts controlled by the promoter predominantly expressed in younger tissues. Subcellular localization study revealed that UGT78H2 was a soluble protein in the nucleus and cytoplasm. These results clarified the bio-function of UGT78H2 and provided a valid approach for substrate selectivity modification in horticultural plants, particularly for sugar donor selectivity. (C) 2020 Elsevier B.V. All rights reserved.

Automated summary

In this study, cloning and functional characterization of a UGT, UGT78H2 in blackberry fruits was reported. The UGT78H2 was found to predominantly glycosylate quercetin and exhibit higher catalytic efficiency towards UDP-galactose.