Dirigent isoflavene-forming PsPTS2: 3D Structure, stereochemical and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea

Pea phytoalexins (–)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2ʹ-dihydroxy-4ʹ,5ʹ-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity towards four possible 7,2ʹ-dihydroxy-4ʹ,5ʹ-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate’s near β-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer’s α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured β-axial OH was closest to Asp51, whereas 4S-isomer’s α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin.