Summary The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate dependent decarboxylating enzyme that uses branched chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched chain amino acids), isotope-labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.
Acetylenic specialized metabolites containing one or more carbon-carbon triple bonds are widespread, being found in fungi, vascular and lower plants, marine sponges and algae, and insects. Plants, moss, and most recently, insects, have been shown to employ an energetically difficult, sequential dehydrogenation mechanism for acetylenic bond formation. Here, we describe the cloning and heterologous expression in yeast of a linoleoyl 12-desaturase (acetylenase) and a bifunctional desaturase with ⌬ 12 -/⌬ 14 -regiospecificity from the Pacific golden chanterelle. The acetylenase gene, which is the first identified from a fungus, is phylogenetically distinct from known plant and fungal desaturases. Together, the bifunctional desaturase and the acetylenase provide the enzymatic activities required to drive oleate through linoleate to crepenynate and the conjugated enyne (14Z)-dehydrocrepenynate, the branchpoint precursors to a major class of acetylenic natural products.The dehydrogenation of oleoyl (18:1 ⌬ 9c ) phosphatidylcholine (PC) to linoleoyl (18:2 ⌬ 9c,12c ) PC in plant and fungal primary metabolism is catalyzed in the endoplasmic reticulum (ER) 2 by the ⌬ 12 desaturase, FAD2, and is central to the functionality of biological membrane systems, cellular signaling, thermal adaptation, and energy storage (1, 2). The oxidative potential of the FAD2 architecture is apparent in the diversification of this non-heme diiron enzyme to a panorama of regioand chemoselective microsomal desaturases that provide most of the functional group diversity in natural acyl lipids, including acetylenic fatty acids (3-5). Bioengineering of seed oil in agronomically important oil-producing plants by controlling flux through seed-specific diverged FAD2s (6, 7) and the manipulation of variant desaturase/acetylenase enzymes may diversify biobased feedstocks. Although acetylenic bond formation is a fundamental transformation, the origin of such highly unsaturated natural products is not well established. In this report we reveal two cooperative multifunctional FAD2 enzymes that produce cis-enyne and trans-alkene building blocks of the naturally occurring acetylenes.Of the acetylenic natural products, the majority are found in plants, moss, and fungi (8). Agrocybin (Fig. 1, 1), the likely origin of fairy rings in grass turf, may promote cell death via apoptosis in trypanosomes (9). Nemotin (2), mycomycin (3), and aryl amide (4) have potent antibiotic activities against nematodes, bacilli (e.g. tuberculosis), and drug-resistant strains of Staphylococcus aureus, respectively (10 -12). (14Z)-Dehydrocrepenynic acid (18:2 ⌬ 9c,12a,14c ) was found to be an inhibitor of bacterial conjugation, suggesting a target for reducing the horizontal gene transfer involved in multi-drug resistance (13). During recent years, a small number of FAD2-diverged genes from plants, moss, and insects have been functionally demonstrated to encode acetylene-forming enzymes or acetylenases (14 -17). Basidiomycete (club) fungi are some of the most prolific producers of ...
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