Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid

Abstract

Understanding how environmental signals regulate production of domoic acid in blooms of Pseudo-nitzschia spp. at a molecular level requires description of the biochemical pathway to this kainoid neurotoxin. Precursor feeding studies have suggested domoic acid arises from the condensation of the C10 isoprenoid geranyl diphosphate with glutamate, but the specific reactions leading to domoic acid from these precursors remain undescribed. Here, we develop a method to derivatize domoic acid with propyl chloroformate that enables gas chromatography-mass spectrometry (GC–MS) analysis to measure incorporation of stable isotopes into domoic acid generated in cultures incubated with isotopically-labeled substrates. We apply this method to demonstrate that both 2H from [1-2H2]geraniol are incorporated into domoic acid, suggesting that the condensation of geranyl diphosphate with an amino group occurs by nucleophilic substitution of the diphosphate rather than by oxidation of geraniol to the aldehyde before reaction with an amino group to form an imine. Ultimately, these and similar studies will facilitate the identification of DA biosynthetic enzymes and genes which will enable the study of how environmental factors regulate DA biosynthesis at the molecular level.