4.7 Article

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

Journal

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 166, Issue -, Pages 277-287

Publisher

ELSEVIER
DOI: 10.1016/j.ijbiomac.2020.10.184

Keywords

UDP-glycosyltransferases; UGT78H2; Substrate specificity

Funding

  1. National Natural Science Foundation of China [31600232]

Ask authors/readers for more resources

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.
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.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.7
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available