Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

Kaushik Ghose,Chris W Kirby,Sylvie J Cloutier,Michael Deyholos,Marva Sweeney-Nixon,Jason Mccallum,Bourlaye Fofana,Raju Datla,Kumarakurubaran Selvaraj

doi:10.1186/1471-2229-14-82

Abstract

BackgroundLignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.ResultsFive UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.ConclusionWe have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Highlights

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta
Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans that
The partial cDNA sequences were analyzed using BLASTx which confirmed the identity of each sequence as belonging to the uridine diphosphate glycosyltransferases (UGTs) family

Summary

Introduction

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. We report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Different types of lignans have been reported in various plant species and include secoisolariciresinol diglucoside (SDG) found mainly in flax (Linum usitatissimum L.) [5,6,7,8,9,10]. Flax seeds are a rich source of SDG lignans that. Glycosylation is one of the key modifications in the secondary metabolite biosynthesis and is catalyzed by carbohydrate active enzymes (CAZymes) which include the superfamily of glycosyltransferases (GTs) [21]. Barvkar et al [27] probed this flax draft genome and reported 137 flax UGTs but did not assign functions to any of these UGTs

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