Structure elucidation and isotopic labeling experiments provide insights into the biosynthesis of three phytochemicals: alkamides, avenacin, and hydroxylated fatty acids

Abstract

Plants are an essential resource providing the world with food, oxygen to breath, and animal habitats. Within each plant, there is an integrated network of pathways that produces phytochemicals either metabolism [1, 2]. Following the discovery of the first specialized metabolite in 1806, the common view has shifted from describing a collection of “by-products” to primary metabolism [3, 4] to the products of a value-added expansion of primary metabolic processes that provides an adaptive advantage to the producing organism. Herein, we report the identification of metabolites for three lipid-related pathways and insights into their origins. The first study was initiated by the serendipitous discovery of unusual fatty acids in the seed oil of Orychophragmus violaceus. Structural analyses together with targeted syntheses of derivatives and molecular fragments revealed two C24 dihydroxy fatty acids identified as 7,18-(OH)2-24:1∆15 and 7,18-(OH)2-24:2∆15,21 that account for ~50% of the seed oil. Evidence indicates that these hydroxylated fatty acids are made via discontinuous elongation, a novel process intermediate between fatty acid and polyketide syntheses. Second, 13C and 2H labeled precursors were used to gain insight into the biosynthetic pathway of alkamides produced by Echinacea. It was determined that the alkamide’s amide moiety is derived from valine and isoleucine, which led to the discovery of the first plant branched-chained amino acid decarboxylase. Separately, the involvement of these atypical acyl chains in alkamide biosynthesis was tested with [2H3] 2E-dodec-, tridec-, and tetradecenoic acids, and their isobutylamides. Analogous CoA derivatives were synthesized to characterize a ligase potentially responsible for the amide bond formation. Finally, to gain insight into the mechanism of a divergent CYP51 enzyme involved in avenacin biosynthesis, epoxy and hydroxylated triterpenes formed by mutant enzymes were identified. Collectively, these studies further the knowledge of the biosynthesis of specialized plant natural products.