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The Biosynthetic Pathway of Major Avenanthramides in Oat

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The Biosynthetic Pathway of Major Avenanthramides in Oat

Zhiyong Li et al. Metabolites.

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.

Keywords: 4-coumarate-CoA ligase; Avena sativa; avenanthramides; caffeoyl-CoA O-methyltransferase; hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
In vitro assays of oat 4CL gene (As4CL1) using the protein expressed in E. coli on three substrates. HPLC analysis of the products on substrate ferulic acid (A), p-coumaric acid (B), and caffeic acid (C).
Figure 2
Figure 2
In vitro assays of oat HHT gene (AsHHT1) using the protein expressed in E. coli. HPLC analysis of the products in the presence of 5-hydroxy-anthranilic acid with p-coumaroyl-CoA (A) and with caffeoyl-CoA (C). The negative controls with boiled AsHHT enzymes were shown in (B,D) with corresponding substrates.
Figure 3
Figure 3
In vitro assays of CCoAOMT using the protein expressed in E. coli on three substrates. HPLC analysis of the products in the presence of S-adenosyl methionine with Avn-C (A), caffeoyl-CoA (B) and caffeic acid (C).
Figure 4
Figure 4
Mutagenesis analysis of the oat CCoAOMT enzyme. (A) Sequence alignment of oat CCoAOMT sequence and related sequences. Conserved residues involved in SAM binding were highlighted in red boxes. Residues involved in divalent binding were highlighted in green boxes. The loop region was between N227 to L250. Mutation site were marked by red *. Changes in the activity of native and mutagenized CCoAOMT proteins on three substrates avenanthramide C (B), caffeoyl-CoA (C), and caffeic acid (D) were calculated using the average of three biological triplicate measurements.
Figure 5
Figure 5
The complete biosynthetic pathway of three major avenanthramides in oat.

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References

    1. Webster F.H., Wood P.J. Oats: Chemistry and Technology. American Association of Cereal Chemists; St. Paul, MN, USA: 2011.
    1. Sadiq-Butt M., Tahir-Nadeem M., Khan M.K.I., Shabir R., Butt M.S. Oat: Unique among the cereals. Eur. J. Nutr. 2008;47:68–79. doi: 10.1007/s00394-008-0698-7. - DOI - PubMed
    1. Singh R., De S., Belkheir A. Avena sativa (oat), a potential neutraceutical and therapeutic agent: An overview. Crit. Rev. Food Sci. Nutr. 2013;53:126–144. doi: 10.1080/10408398.2010.526725. - DOI - PubMed
    1. Adom K.K., Liu R.H. Antioxidant activity of grains. J. Agric. Food Chem. 2002;50:6182–6187. doi: 10.1021/jf0205099. - DOI - PubMed
    1. Liu L., Zubik L., Colllins W., Marko M., Meydani M. The antiatherogenic potential of oat phenolic compounds. Atherosclerosis. 2004;175:39–49. doi: 10.1016/j.atherosclerosis.2004.01.044. - DOI - PubMed
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