Formation of a new class of oxylipins from N‐acyl(ethanol)amines by the lipoxygenase pathway

Abstract

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both a-N-linolenoylethanolamine and g-N-linolenoylethanolamine (18:3), as well as a-N-linolenoylamine and g-N-linolenoylamine were converted into their (13S )-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of a-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z )-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z )-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S )-hydroperoxy-a-N-linolenoylethanolamine and the (13S )-hydroperoxide of linoleic acid and its ethanolamine derivative. a-Ketols and g-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S )-hydroperoxy-a-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.

Oxygenated Metabolites of Anandamide and 2-Arachidonoylglycerol: Conformational Analysis and Interaction with Cannabinoid Receptors, Membrane Transporter, and Fatty Acid Amide Hydrolase

“…28,[44][45][46][47][48] The spectra were in accordance with their chemical structures, as previously reported. 28,44,47,48 Each lipoxygenase was incubated with LEA, AEA (Cayman Chemicals), 3-AG, or 1-AG (1 U enzyme lipoxygenase per 3 µmol substrate) in 100 mM sodium borate buffer (pH 9 for soybean and pH 7 for barley grain). AEA was incubated with bovine polymorphonuclear leukocytes in phosphate-buffered saline (pH 7.4) to produce 12S-HAEA.…”

N-Acylethanolamines Are Metabolized by Lipoxygenase and Amidohydrolase in Competing Pathways during Cottonseed Imbibition

“…4C). Predictable fragmentation ions including the molecular ion [M ϩ ] (m/z 573) were clearly identifiable, and spectra were comparable with those recorded in previous studies (Van der Stelt et al, 2000). Only ␣-ketols originating from 13-hydroperoxy NAE (18:2) were detected, indicating that imbibed cottonseeds contained both a 13-LOX and 13-AOS that metabolized NAE 18:2.…”

“…Recent evidence demonstrated that purified plant LOX, AOS, and HPO lyase could metabolize synthetic NAEs to generate novel oxylipins (Van der Stelt et al, 2000), raising the possibility that plants might catalyze these reactions in vivo. Here, we provide several lines of evidence that support this concept and highlight seed imbibition and germination as a period intensely involved in formation of these metabolites.…”

“…The hydroperoxides of NAE 18:2 and NAE 18:3 could subsequently be converted by alfalfa (Medicago sativa) hydroperoxide (HPO) lyase and flax (Linum usitatissimum) seed allene oxide synthase (AOS) into novel oxylipins (Van der Stelt et al, 2000). N-Arachidonoylethanolamine (anandamide), the mammalian neurotransmitter, could be converted into hydroperoxy NAE by purified 5-LOX from barley (Hordeum vulgare) and tomato (Lycopersicon esculentum; Van Zadelhoff et al, 1998).…”

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…Analyses of the specificity of the tea leave HPL, a 13-HPL, and our results indicated that the absolute position of the diene system in relation to the methylene end and not to the carboxyl end is an important factor for the substrate specificity. Nevertheless the carboxy group plays also a role, since methylation reduces often the activity whereas to some extent an acid-amine is a better substrate (van der Stelt et al, 2000;Kandzia et al, 2003). An influence of the pH is also discussed in respect to amount of carboxylate anion in relation to the undissociated carboxyl group.…”