Abstract
Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.
Highlights
The human food market for oat is increasingly important as the consumers gradually recognize its health benefits [1]
The biosynthesis of hydroxycinnamoyl-CoAs is generally believed to be catalyzed by 4-coumarate-CoA ligase (4CL)
To identify genes encoding this enzyme in oat, an Arabidopsis thaliana 4CL was used as a query to search a transcriptome database of oat developing seeds [24]
Summary
The human food market for oat is increasingly important as the consumers gradually recognize its health benefits [1]. HCT is a sub-family of well-conserved enzymes among land plants participating in the biosynthesis of lignins and flavonoids that function by acylating a wide range of aroyl group-containing substrates to appropriate acceptors [13,16,17,18,19]. Acyl donors such as hydroxycinnamoyl-CoA and derivatives, precursors for the biosynthesis of avenanthramides are probably diverged from the phenylpropanoid pathway and their biosynthesis may require 4-coumarate: CoA ligase (4CL) for the activation of aroyl group-containing substrates to the corresponding thioesters for the subsequent condensation process [20,21,22]. Elucidation of the biosynthesis of the major avenanthramides would facilitate breeding efforts to improve this important nutritional trait by functional DNA markers-assistant breeding in oat
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