Identification of a Bifunctional Maize C- and O-Glucosyltransferase

María Lorena Falcone Ferreyra,Eduardo Rodriguez,María Isabel Casas,Guillermo Labadie,Erich Grotewold,Paula Casati

doi:10.1074/jbc.m113.510040

Abstract

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.

Highlights

Plant uridine diphosphate (UDP)-glycosyltransferases add sugars to acceptors like flavonoids either via hydroxyls (O-linkage) or carbons (C-linkage)
We have previously demonstrated that a flavanone 2-hydroxylase (ZmF2H1), CYP93G5, converts flavanones into the corresponding 2-hydroxyflavanones [25], which are proposed to serve as substrates for C-glycosylation, followed by dehydration as has been described in other grasses [9, 26, 27]
Glycosylation is an important step in flavonoid biosynthesis that contributes to flavonoid stability, solubility, storage, and biological activity changes [3]

Summary

Background

Plant UDP-glycosyltransferases add sugars to acceptors like flavonoids either via hydroxyls (O-linkage) or carbons (C-linkage). Maysin, the C-glycosyl flavone predominant in silk tissues of some maize varieties, is a natural insecticide against the corn earworm Helicoverpa zea [15, 16], whereas C-glycosyl flavonoids identified in cucumber leaves act as phytoalexins in defense against powdery mildew fungi [17, 18]. UGT708A6 can accept flavanones as substrates to form O-glycosidated products These dual activities were confirmed by both in vivo bioconversion assays and in vitro assays with the recombinant protein, revealing that UGT708A6 is a bifunctional enzyme with the ability to form both C-glycoside and O-glycoside derivatives using as acceptors 2-hydroxyflavanones and flavanones, respectively

EXPERIMENTAL PROCEDURES

RESULTS

E eriodictyol 7-O-glucoside

DISCUSSION

Findings

B UGT708A6