Plant P5C reductase as a new target for aminomethylenebisphosphonates

A series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as potential inhibitors of delta1-pyrroline-5-carboxylate reductase (EC 1.5.1.2), the enzyme that catalyzes the last step in proline biosynthesis, partially purified from Arabidopsis thaliana suspension cultured cells. At millimolar concentrations, three compounds out of 26 were found to interfere with the catalytic mechanism. One of them, namely, 3,5-dichloropyridyl-aminomethylenebisphosphonic acid, retained such inhibitory activity in the micromolar range. Kinetic analyses ruled out the possibility that the inhibition could simply rely upon the chelating properties of bisphosphonates and showed mechanisms of a noncompetitive type against NADH and an uncompetitive type against delta1-pyrroline-5-carboxylic acid, with KI values of 199 +/- 6 and 10.3 +/- 1.5 microM, respectively. A computer-aided docking analysis, performed on the basis of the crystal structure of the enzyme from Streptococcus pyogenes, suggested that this phosphonate may interact with amino acid residues near the binding site of delta1-pyrroline-5-carboxylic acid, thus blocking the substrate in a pocket and preventing its interaction with NADH. Because in higher plants the step catalyzed by delta1-pyrroline-5-carboxylate reductase is shared by all pathways leading to proline synthesis, such a compound may represent a lead structure to be exploited for the design of new substances endowed with herbicidal activity.