Identification of a Flavonoid Glucosyltransferase Involved in 7-OH Site Glycosylation in Tea plants (Camellia sinensis)

Xinlong Dai,Yingling Wu,Yajun Liu,Peiqiang Wang,Juhua Zhuang,Xiaolan Jiang,Tao Xia,Guifu Zhao,Liping Gao

doi:10.1038/s41598-017-06453-z

Abstract

Flavonol glycosides, which are often converted from aglycones in a process catalyzed by UDP-glycosyltransferases (UGTs), play an important role for the health of plants and animals. In the present study, a gene encoding a flavonoid 7-O-glycosyltransferase (CsUGT75L12) was identified in tea plants. Recombinant CsUGT75L12 protein displayed glycosyltransferase activity on the 7-OH position of multiple phenolic compounds. In relative comparison to wild-type seeds, the levels of flavonol-glucosides increased in Arabidopsis seeds overexpressing CsUGT75L12. In order to determine the key amino acid residues responsible for the catalytic activity of the protein, a series of site-directed mutagenesis and enzymatic assays were performed based on the 3D structural modeling and docking analyses. These results suggested that residue Q54 is a double binding site that functions as both a sugar receptor and donor. Residues H56 and T151, corresponding to the basic active residues H20 and D119 of VvGT1, were not irreplaceable for CsUGT75L12. In addition, residues Y182, S223, P238, T239, and F240 were demonstrated to be responsible for a ‘reversed’ sugar receptor binding model. The results of single and triple substitutions confirmed that the function of residues P238, T239, and F240 may substitute or compensate with each other for the flavonoid 7-O-glycosyltransferase activity.

Highlights

Since only a few of the genes responsible for the biosynthesis of glycosylated phenolic compounds have been identified in tea plants, our understanding is limited with respect to synthesized compounds and their respective physiological roles in tea plants
Among 91 recombinant UGTs analyzed in Arabidopsis thaliana, 29 exhibited significant catalytic activity toward quercetin; of which eleven catalyzed the glucosylation at the 3-OH position
The key amino acids involved in the catalytic mechanism of flavonoid 3-O-glycosyltransferase were illustrated by solved crystal structures of the plant UGTs, UGT78G1 and UGT85H2 from M. truncatula and VvGT1 from V. v inifera[13, 24, 25]

Summary

Introduction

Since only a few of the genes responsible for the biosynthesis of glycosylated phenolic compounds have been identified in tea plants, our understanding is limited with respect to synthesized compounds and their respective physiological roles in tea plants. Β-glucogallin (βG), a glucose ester of gallic acid, acts as an efficient acyl donor during the biosynthesis of galloylated catechins[6] Both CsUGT78A14 and CsUGT78A15 were identified as glycosyltransferases that catalyze the glycosylation of flavonoids at the 3-OH position using UDP-glucose and UDP-galactose as sugar donors[5]. While the sequences of a large number of plant UGT genes are available in public databases, only a few crystal structures of UGT proteins have been reported[13, 22,23,24,25]. The key amino acids involved in the catalytic mechanism of flavonoid 3-O-glycosyltransferase were illustrated by solved crystal structures of the plant UGTs, UGT78G1 and UGT85H2 from M. truncatula and VvGT1 from V. v inifera[13, 24, 25]. Little is known regarding to the amino acids that play key functional roles in flavonoid 7-O-glycosyltransferase activity

Methods

Results

Conclusion