(+)-Pisatin biosynthesis: From (-) enantiomeric intermediates via an achiral 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene

(+)-Pisatin, produced by peas (Pisum sativum L.), is an isoflavonoid derivative belonging to the pterocarpan family. It was the first chemically identified phytoalexin, and subsequent research has demonstrated that most legumes produce pterocarpans with the opposite stereochemistry. Studies on the biosynthesis of (+)-pisatin have shown that (-) enantiomeric compounds are intermediates in (+)-pisatin synthesis. However, the steps from the (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] intermediate to (+)-6a-hydroxymaackiain intermediate are undetermined. Chemical reduction of ()sophorol using sodium borohydride (NaBH4) produced two isomers of (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI] with optimal UV absorbance at 299.3 and 300.5 nm, respectively. In contrast, enzymatic reduction of (-)-sophorol by the pea enzyme sophorol reductase (SOR) produced only the 299.3 nm (-)-DMDI isomer. Proton nuclear magnetic resonance (H-1 NMR) analysis of the 299.3 nm (-)-DMDI isomer demonstrated that this isomer had the same NMR spectrum as previously reported for cis-isoflavanol isomers, indicating that cis-(-)-DMDI is an intermediate in (+)-pisatin biosynthesis. Enzyme assays using protein extracts from pea tissue treated with CuC12 as an elicitor converted the cis-(-)-DMDI isomer into an achiral isoflavene, 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and the trans-(-)-DMDI isomer was not metabolized by the same protein preparation. A comparison of the enzyme activities on cis-(-)-DMDI with protein preparations from elicited tissue versus non-elicited tissue showed a threefold increase in the amount of activity in the proteins from the elicited tissue. Proteins from the elicited tissues of alfalfa, bean, and chickpea converted cis-(-)-DMDI into either (-)-maackiain and/or (-)-sophorol, while proteins from the elicited tissues of broccoli and pepper produced no detectable product. These results are consistent with the involvement of cis-(-)-DMDI and the achiral DMDIF as intermediates in (+)-pisatin biosynthesis. (C) 2013 Elsevier Ltd. All rights reserved.