The structural model reveals THNR to belong to the family of short chain dehydrogenases. Despite the diversity of the chemical reactions catalyzed by this family of enzymes, their tertiary structures are very similar. In particular THNR has many amino acid sequence identities, and thus most probably high structural similarities, to enzymes involved in fungal aflatoxin synthesis. The structure of THNR in complex with NADPH and tricyclazole provides new insights into the structural basis of inhibitor binding. This new information may aid in the design of new inhibitors for rice crop protection.

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 complexes. The partition ratio of U7278 and a physiological substrate (scytalone) was 95:1 and unchanged on varying 3HNR.NADP+/3HNR(unliganded), which is also consistent with the ordered mechanism. The pH dependence of the hydride transfer rate from U7278 to NADP+ was measured, as was the pH dependence of Kcat/K(m)(NADP+). Hydride transfer had a pH dependence which suggests a single deprotonated residue (pKa = 6.0) is required for catalysis. Khyd, the rate constant for hydride transfer, was 9-fold larger than Kcat with U7278 as a substrate. A burst in the pre-steady-state suggests that release of one or both of the products is rate limiting to Kcat at pH 7.0. The pH dependence of Kcat/K(m)(NADP+) indicates a requirement for a single deprotonated group and this ionization is assigned to the 2' phosphate of NADP+. 3HNR was found to be 800-fold more specific for NADP+ relative to NAD+. Analysis of sequence and structure [Anderson, A., Jordan, D. B., Schneider. G., & Lindqvist. Y. (1996) Structure 4, 1161-1170] reveals that 3HNR is a member of the short-chain dehydrogenase superfamily of enzymes.

The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10-40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal 'X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.

The crystal structures of apo-l,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea and a binary complex of the enzyme with NADPH have been determined to 2.8 A resolution. In both cases, the overall structure is preserved compared to the structure of the ternary complex of the enzyme with NADPH and an active site inhibitor. No electron density for the helix-loop-helix region comprising residues 214-244 is observed indicating structural disorder in this part of the apoenzyme and the binary complex. In the ternary complex, this region is in contact with NADPH and the inhibitor and closes off the active site. The observed increase in flexibility in the absence of the inhibitor indicates that this region acts as a lid which closes the active site upon binding of the inhibitor and, possibly the substrate, 1,3,8-trihydroxy naphthalene.