Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

Abstract

Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (-)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used (18)O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under (18)O(2), the product was labeled with 99% efficiency; no label was incorporated by metabolism in H(2) (18)O. Pisatin synthesized by pea pods in the presence of (18)O(2) or H(2) (18)O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H(2)O and not from O(2). Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.