Abstract
An isoflavan vestitol is a phytoalexin of the Lotus spp. including a model legume Lotus japonicus. Despite of extensive studies on isoflavonoid metabolism, detailed mechanism of the isoflavan biosynthesis has been unclear. In this study, homologous sequences (PTR1 and PTR2) of phenylcoumaran benzylic ether reductase (PCBER), an enzyme of lignan pathway catalyzing an analogous reaction to pterocarpan reduction, were selected from a L. japonicus EST library, and substrate specificity of the proteins was examined. At the amino acid levels, PTRs are 60% identical with both PCBER and isoflavone reductase. Crude extracts of E. coli expressing PTRs catalyzed the formation of vestitol from a pterocarpan (-)-medicarpin and NADPH. Lignans and 2'-hydroxyformononetin were not used as substrates. Thus, PTRs showed pterocarpan reductase activity. RT-PCR analysis using L. japonicus seedlings displayed the constitutive accumulations of PTR mRNAs with or without reduced glutathione-treatment. Further characterization of the enzymes is in progress.
