Abstract
While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.
Highlights
Peroxygenase is a hydroperoxide-dependent oxygenase that catalyzes the transfer of one oxygen atom from a hydroperoxide to a substrate that is oxidized (Blee et al, 1993)
Identification of cDNAs encoding fatty acid peroxygenase and lipoxygenases from A. sativa To identify a cDNA encoding fatty acid peroxygenase from oat, A. sativa EST databases prepared from leaves, roots and developing seeds at different stages that are publically available from NCBI were searched using Arabidopsis ATS1, an embryo-specific caleosin (Nuccio and Thomas 1999) as a query sequence
The results showed that AsPXG1 could only use free fatty acid and fatty acid methyl esters as substrates; whereas phospholipid and acyl-CoA were not effective
Summary
Peroxygenase is a hydroperoxide-dependent oxygenase that catalyzes the transfer of one oxygen atom from a hydroperoxide to a substrate that is oxidized (Blee et al, 1993). Peroxygenase activity was first detected in oat (Avena sativa L.) when 9- and 13-hydroperoxyoctadecadienoic acids were reduced to the corresponding alcohols and 9,10-epoxy-13hydroxyoctadecenoic acids or 12,13-epoxy-9-hydroxyoctadecenoic acids was formed (Heimann and Schreier 1970;Heimann and Dresen 1973) This reaction was believed to be catalyzed by a “lipoperoxidase activity” or “hydroperoxide isomerase”. In 1977, peroxygenase was first defined by a labeling study using pea microsomes (Ishimaru and Yamazaki 1977) as hydroperoxide-dependent monooxygenase, which differs from typical cytochrome P450 monooxygenase in the oxygen donor Since this type of enzyme activity has been detected in various microorganisms and plants species (Blee et al, 1993;Matsunaga and Shiro 2004). The peroxygenase activity is strictly dependent on the binding of calcium and ferric-heme (Hanano et al, 2006)
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