Trihydroxynaphthalene Reductase from Magnaporthe grisea: Realization of an Active Center Inhibitor and Elucidation of the Kinetic Mechanism

Abstract

Active trihydroxynaphthalene reductase (3HNR) is essential for the biosynthesis of fungal melanin by Magnaporthe grisea and is a focus of inhibitor design studies directed toward control of blast disease in rice. Tricyclazole, a preventative fungicide against rice blast, has been previously characterized as inhibiting 3HNR noncompetitively [Viviani, F., Vors, J. P., Gaudry, M., & Marquet, A. (1993) Bull. Soc. Chem. Fr. 136, 395-404] with respect to its naphthol substrate. Our steady-state kinetic and fluorescence titration studies show that instead the inhibitor binds competitively with respect to the naphthol substrate and that it binds to 3HNR forms with the preferences 3HNR.NADPH > 3HNR.NADP+ > 3HNR (unliganded): Kt = 15 nM, 0.56 microM, and Kd = 8.5 microM, respectively. Analysis of the frontier molecular orbitals of tricyclazole and NADP(H) provides a basis for the affinity differences of tricyclazole for 3HNR.NADP(H) enzyme forms. Fluorescence titrations show that NADPH and naphthol substrates form binary complexes with 3HNR [Kd(NADP+) = 38 microM and Kd(U7278, an alternate naphthol-like substrate) = 220 microM]. However, the overwhelmingly preferred order of productive binding is NADPH followed by naphthol substrate, as shown by the uncompetitive inhibition of 3HNR by tricyclazole with respect to NADPH. Consistent with this mechanism, the K(m)'s for the naphthol substrates U7278 and scytalone (5 and 6 microM, respectively) are much lower than the Kd's of the binary com…