Molecular cloning and biochemical characterization of isoflav-3-ene synthase, a key enzyme of the biosyntheses of (+)-pisatin and coumestrol

Most leguminous plants produce (-)-type enantiomers of pterocarpans as the phytoalexin, but pea (Pisum sativum L.) produces the opposite stereoisomer of pterocarpan, (+)-pisatin. Biosynthesis of (-)-pterocarpan skeleton is completely characterized at the molecular level, and pterocarpan synthase (PTS), a dirigent (DIR) domain-containing protein, participates in the last dehydration reaction. Similarly, isoflav-3-ene, a precursor of (+)-pisatin, is likely to be biosynthesized by the DIR-mediated dehydration reaction; however the biosynthesis is still unknown. In the present study, we screened PTS homologs based on RNA sequence data from (+)-pisatin-producing pea seedlings and demonstrated that one of the candidates encodes isoflav-3-ene synthase (I3S). Real-time PCR analysis revealed that transcripts of I3S, in addition to other genes involved in the (+)-pisatin pathway, transiently accumulated in pea upon elicitation prior to the maximum accumulation of (+)-pisatin. I3S orthologs were also found in soybean and Lotus japonicus that are not known to accumulate (+)-pterocarpan, and the catalytic function of gene products was verified to be I3S by the in vitro enzyme assay. Incubation of the crude extract of elicited soybean cells with isoflav-3-ene yielded coumestrol, suggesting that isoflav-3-ene is a precursor of coumestrol biosynthesis in soybean.