Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins

Amorn Owatworakit,Belinda Townsend,Thomas Louveau,Helen Jenner,Martin Rejzek,Richard K Hughes,Gerhard Saalbach,Xiaoquan Qi,Saleha Bakht,Abhijeet Deb Roy,Sam T Mugford,Rebecca J.M Goss,Robert A Field,Anne Osbourn

doi:10.1074/jbc.m112.426155

Amorn Owatworakit, Belinda Townsend + Show 12 more

Open Access

https://doi.org/10.1074/jbc.m112.426155

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Abstract

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

Highlights

Glycosyltransferases (GTs) have important functions in plant secondary metabolism
The UGT74H6 and UGT74H7 proteins were not detected in any of the protein preparations from oat. These results suggest that of the three enzymes, UGT74H5 is the major GT present in protein preparations derived from oat root tips
Three avenacin pathway enzymes have been identified. These include those encoded by Sad1 (␤-amyrin synthase) [14] and Sad2 [13]

Summary

Background

Glycosyltransferases (GTs) have important functions in plant secondary metabolism. Results: A gene encoding an N-methylanthranilic acid O-glucosyltransferase forms part of a biosynthetic cluster for the synthesis of acylated defense compounds in oat. We identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. We identify a gene predicted to encode a family 1 GT gene belonging to the UGT74 subfamily that forms part of a metabolic gene cluster for avenacin synthesis (UGT74H5) This gene is co-expressed with other previously characterized genes in the avenacin cluster, suggesting that UGT74H5 is required for avenacin synthesis. Our biochemical analysis, coupled with investigation of the distribution of these enzymes in oat roots, indicates that UGT74H5 is likely to be key for the synthesis of the major avenacin, A-1, whereas UGT74H6 may be required for the synthesis of other avenacins that are acylated with benzoic acid

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