MOLECULAR-CLONING OF ISOFLAVONE REDUCTASE FROM PEA (PISUM-SATIVUM L) - EVIDENCE FOR A 3R-ISOFLAVANONE INTERMEDIATE IN (-PISATIN BIOSYNTHESIS())

Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isofl avanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea (Pisum sativum) was isolated using the po lymerase chain reaction and expressed in Escherichia coli. Analysis of circular dichroism (CD) spectra of the reduction product sophorol obt ained using the recombinant enzyme indicated that the isoflavanone pos sessed the 3R stereochemistry, in contrast to previous reports indicat ing a 3S-isoflavanone as the product of the pea IFR. Analysis of CD sp ectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl2-trea ted seedlings, consistent with the appearance of IFR enzyme activity a nd pisatin accumulation. (C) 1994 Academie Press, Inc.