Kinetic Study of Coniferyl Alcohol Radical Binding to the (+)-Pinoresinol Forming Dirigent Protein

Abstract

An essential step in lignan and lignin formation in planta is one electron oxidation of (E)-coniferyl alcohol (CA) to generate the radical intermediate (CA.), which can then undergo directed radical-radical couplings in vivo. For lignan formation in vitro and in vivo, stereoselective coupling of CA. only occurs to afford (+)-pinoresinol in the additional presence of (+)-pinoresinol forming dirigent protein (DP). Presented herein is a kinetic and thermodynamic study which reveals the central mechanistic details of the coupling process involved in DP-mediated coupling. DP activity was maximal between pH 4.25 and pH 6.0, with activity being maintained at temperatures below 33 °C. Equilibrium binding assays revealed that coniferyl alcohol was only weakly bound to the DP, with a KD of 370 ± 65 μM. On the other hand, the enantiomeric excess of (+)-pinoresinol formed was dependent on both DP concentration and rate of CA oxidation and, thus, on apparent steady-state [CA.]. The data obtained could best be explained using a kinetic model where radical-radical coupling via DP competes with that occurring in open solution. Using this model, an apparent KM of about 10 nM was estimated from the saturation behavior of (+)-pinoresinol formation with respect to apparent steady-state [CA.]. These data strongly suggest that CA., rather than CA, is the substrate for DP, in agreement with earlier predictions. A mechanism of directed radical-radical coupling, where two coniferyl alcohol radical substrates are bound per protein dimer, is proposed.