RESUMEN
Rice Blast Disease, caused by the fungus Pyricularia oryzae, is one of the most important diseases of rice. Several enzymes in the melanin biosynthetic pathway have proven to be valuable targets for development of rice blast fungicides. In particular, inhibitors of trihydroxynaphthalene reductase (3HNR), which catalyzes the conversion of trihydroxynaphthalene to vermelone, have yielded commercially useful rice fungicides. The X-ray structure of 3HNR has been published recently, presenting an opportunity to use this information in the de novo design of novel 3HNR inhibitors that may exhibit useful rice blast activity. We used the LeapFrog program to develop a docking model for interaction of ligands with the active site of THNR. The final model gave a good correlation between calculated binding energy and log Ki and was used to design novel ligands and score compounds for synthesis. Using this as a tool, we synthesized inhibitors in the nanomolar range and also developed several inhibitors that did not conform to the properties of the THNR active site. Leapfrog was able to locate a previously unrecognized binding pocket that could accommodate these otherwise anomalous regions of structure.
Asunto(s)
Ascomicetos/enzimología , Inhibidores Enzimáticos/síntesis química , Proteínas Fúngicas , Fungicidas Industriales/síntesis química , Oryza/microbiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Fungicidas Industriales/química , Modelos Moleculares , Oxidorreductasas/química , Conformación ProteicaRESUMEN
BACKGROUND: Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. RESULTS: Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 A (pyroquilon), 2.0 A (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 A (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 in each of the three structures likely serves to stabilize the cationic form of the residue for participation in catalysis. CONCLUSIONS: The pro S hydrogen of NADPH (which is transferred as a hydride to the enzyme's naphthol substrates) is directed toward the carbonyl carbon of the inhibitors that mimic an intermediate along the reaction coordinate. Modeling tetrahydroxynaphthalene and trihydroxynaphthalene in the active site shows steric and electrostatic repulsion between the extra hydroxyl oxygen of the former substrate and the sulfur atom of Met-283 (the C-terminal residue), which accounts, in part, for the 4-fold greater substrate specificity for trihydroxynaphthalene over tetrahydroxynaphthalene.
Asunto(s)
Proteínas Fúngicas , Magnaporthe/enzimología , Melaninas/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/química , Antifúngicos/farmacología , Sitios de Unión , Catálisis , Cationes , Cristalografía por Rayos X , Hidrógeno/química , Melaninas/química , Modelos Químicos , Modelos Moleculares , NADP/química , NADP/metabolismo , Naftalenos/química , Oxidorreductasas/metabolismo , Oxígeno/química , Unión Proteica , Especificidad por SustratoRESUMEN
We explore the use of site-directed mutations of scytalone dehydratase to study inhibitor binding interactions. The enzyme is the physiological target of new fungicides and the subject of inhibitor design and optimization. X-ray structures show that potent inhibitors (K(i)'s approximately 10(-)(11) M) interact mostly with 11 amino acid side chains and, in some cases, with a single backbone amide. Fifteen site-directed mutants of the 11 enzyme residues were prepared to disrupt enzyme-inhibitor interactions, and inhibition constants for 13 inhibitors were determined to assess changes in binding potencies. The results indicate that two of the six hydrogen bonds (always present in X-ray structures of native enzyme-inhibitor complexes) are not important for inhibitor binding. The other four hydrogen bonds are important for inhibitor binding, and the strength of the individual bonds is inhibitor-dependent. Inhibitor atoms remote from the hydrogen bonds influence their strength, presumably by effecting small changes in inhibitor orientation. Several hydrophobic amino acid residues are important recognition elements for lipophilic inhibitor functionalities, which is fully consistent with X-ray structures determined from crystals of enzyme-inhibitor complexes grown at neutral pH but not with those determined from crystals grown under acidic conditions. This study of mutant enzymes complements insights from X-ray structures and structure-activity relationships of the wild-type enzyme for refining views of inhibitor recognition.
Asunto(s)
Hidroliasas/antagonistas & inhibidores , Hidroliasas/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Hidroliasas/química , Enlace de Hidrógeno , Cinética , Magnaporthe/enzimología , Magnaporthe/genética , Modelos Químicos , Mutagénesis Sitio-DirigidaRESUMEN
A new fungicide lead has been identified by enzyme screening of a focused combinatorial library. The lead compound 4, a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic activity. The X-ray crystal structure of the enzyme-inhibitor complex and an appreciation for the relationship between physical properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen-methyl group combination was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF3 with methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while significantly increasing systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for outdoor testing in flooded plots which simulate rice paddies.
Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Fungicidas Industriales/farmacología , Melaninas/antagonistas & inhibidores , Amidas/química , Ciclobutanos/química , Inhibidores Enzimáticos/química , Estudios de Evaluación como Asunto , Fungicidas Industriales/química , Hidroliasas/antagonistas & inhibidores , Melaninas/biosíntesis , Estructura Molecular , Análisis EspectralRESUMEN
[reaction--see text] The major conformation of scytalone has an envelope shape with C3 forming the flap and the C3 hydroxyl in the equatorial position as determined by quantum mechanical calculations and corroborated by NMR. The C2 axial pro-R is slower to exchange with solvent than the equatorial pro-S hydrogen. Modeling the transition state for enolate formation points to a deprotonation through the flipped envelope conformation in which the C3-hydroxyl and the C2 pro-S hydrogen are axial.
Asunto(s)
Naftoles/química , Antifúngicos/química , Antifúngicos/metabolismo , Sitios de Unión , Catálisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Hidroliasas/antagonistas & inhibidores , Conformación Molecular , Naftoles/metabolismo , Resonancia Magnética Nuclear Biomolecular , Solventes/metabolismo , Termodinámica , Agua/metabolismoRESUMEN
Compounds that control rice blast, but not other crop diseases, were selected for testing as inhibitors of trihydroxynaphthalene reductase of the fungal melanin biosynthetic pathway. A potent inhibitor of the enzyme (2) (Ki = 25 nM) was identified. An X-ray structure of the enzyme-NADPH-2 complex was determined at 2.1 A resolution.
Asunto(s)
Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas , Hongos/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Enfermedades de las Plantas/microbiología , Antifúngicos/química , Bases de Datos Factuales , Inhibidores Enzimáticos/química , Melaninas/biosíntesis , Conformación Molecular , NADP/química , Oryza , Difracción de Rayos XRESUMEN
In D(2)O, scytalone exchanges its two C2 hydrogen atoms for deuterium atoms at different rates. At pD 7.0 and 25 degrees C, half-lives for the exchanges are 0.8 and 10 days for the pro-S and pro-R hydrogens, respectively. The differential exchange rates allow for the preparation of multiple scytalone samples (through incubation of scytalone in D(2)O and then back exchanging with H(2)O) having differential levels of deuterium enrichment at the C2 pro-S and pro-R positions. From these samples, the stereochemical preference for hydrogen abstraction during the dehydration reaction mediated by the enzyme scytalone dehydratase was determined. At pH 7. 0, deuterium at the pro-S position has little effect on enzyme catalysis, whereas deuterium at the pro-R position produces kinetic isotope effects of 2.3 (25 degrees C), 5.1 (25 degrees C), and 6.7 (6.8 degrees C) on k(cat), k(cat)/K(m), and the single-turnover rate, respectively. The results are fully consistent with the enzyme catalyzing a syn elimination through an E1cb-like mechanism. The syn elimination is compatible with the interactions realized between a scytalone boat conformation and key active site residues as modeled from multiple X-ray crystal structures of the enzyme in complexes with inhibitors.
Asunto(s)
Proteínas Fúngicas/química , Hidroliasas/química , Naftoles/química , Sitios de Unión , Catálisis , Óxido de Deuterio/química , Hidrógeno/química , Radical Hidroxilo/química , Cinética , Magnaporthe/enzimología , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , EstereoisomerismoRESUMEN
Two water molecules reside between inhibitors and active site residues of scytalone dehydratase. A molecular dynamics study is consistent with one water molecule binding less tightly than the other. Inhibitor binding studies with site-directed mutants indicate that the hydrogen bonding network around the less mobile water molecule contributes much greater binding energy than that around the more mobile one.
Asunto(s)
Hidroliasas/metabolismo , Agua/metabolismo , Sitios de Unión , Simulación por Computador , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Hidroliasas/antagonistas & inhibidores , Hidroliasas/química , Hidroliasas/genética , Cinética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Salicilamidas/metabolismo , Termodinámica , Agua/químicaRESUMEN
A method for the determination of inhibition constants for catalytically-debilitated mutant enzymes is described. The inhibitor is partitioned between the mutant and wild-type enzymes. Catalytic rates of the wild-type enzyme are used as the signal of inhibitor binding to the mutant enzyme. The method is validated with scytalone dehydratase, the Y50F mutant, and a potent inhibitor. The K(i) value for Y50F determined by this method is 0.49 +/- 0.10 nM. The K(i) value determined using the Y50F catalytic report for inhibitor binding in the absence of wild-type enzyme is 0.20 +/- 0.030 nM. The wild-type enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inhibitor (suggested by X-ray crystallography) is weak. The method is most useful when the catalytic activity of the wild-type enzyme is the most sensitive report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity.
Asunto(s)
Benzopiranos/farmacología , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Hidroliasas/química , Sustitución de Aminoácidos , Catálisis , Dominio Catalítico , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/químicaRESUMEN
Five X-ray crystal structures of scytalone dehydratase complexed with different inhibitors have delineated conformationally flexible regions of the binding pocket. This information was used for the design and synthesis of a norephedrine-derived cyanoacetamide class of inhibitors leading to potent fungicides.
Asunto(s)
Antifúngicos/síntesis química , Hidroliasas/antagonistas & inhibidores , Fenilpropanolamina/análogos & derivados , Fenilpropanolamina/síntesis química , Cristalografía por Rayos X , Cinética , Modelos Químicos , Modelos Moleculares , Enfermedades de las PlantasRESUMEN
Insights gained from a crystal structure of scytalone dehydratase led to the design of carboxamide inhibitors with a phenoxypropyl group substituted on the nitrogen atom Potent enzyme inhibitors were synthesized around this motif, the best of which provided excellent control of rice blast disease in greenhouse assays and outdoor field trials.
Asunto(s)
Antifúngicos/síntesis química , Hidroliasas/antagonistas & inhibidores , Cristalografía por Rayos X , Ciclopropanos/química , Diseño de Fármacos , Cinética , Modelos Químicos , Modelos Moleculares , Enfermedades de las Plantas , Tirfostinos/químicaRESUMEN
Scytalone dehydratase is a molecular target of inhibitor design efforts aimed at preventing the fungal disease caused by Magnaporthe grisea. A method for cocrystallization of enzyme with inhibitors at neutral pH has produced several crystal structures of enzyme-inhibitor complexes at resolutions ranging from 1.5 to 2.2 A. Four high resolution structures of different enzyme-inhibitor complexes are described. In contrast to the original X-ray structure of the enzyme, the four new structures have well-defined electron density for the loop region comprising residues 115-119 and a different conformation between residues 154 and 160. The structure of the enzyme complex with an aminoquinazoline inhibitor showed that the inhibitor is in a position to form a hydrogen bond with the amide of the Asn131 side chain and with two water molecules in a fashion similar to the salicylamide inhibitor in the original structure, thus confirming design principles. The aminoquinazoline structure also allows for a more confident assignment of donors and acceptors in the hydrogen bonding network. The structures of the enzyme complexes with two dichlorocyclopropane carboxamide inhibitors showed the two chlorine atoms nearly in plane with the amide side chain of Asn131. The positions of Phe53 and Phe158 are significantly altered in the new structures in comparison to the two structures obtained from crystals grown at acidic pH. The multiple structures help define the mobility of active site amino acids critical for catalysis and inhibitor binding.
Asunto(s)
Inhibidores Enzimáticos/química , Hidroliasas/antagonistas & inhibidores , Secuencia de Bases , Sitios de Unión , Calcio/química , Cristalografía por Rayos X , Cartilla de ADN , Hidroliasas/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Conformación ProteicaRESUMEN
On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.
Asunto(s)
Hidroliasas/química , Hidroliasas/metabolismo , Benzopiranos/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Hidroliasas/genética , Concentración de Iones de Hidrógeno , Cinética , Magnaporthe/enzimología , Mutagénesis Sitio-Dirigida , Naftoles/química , Conformación Proteica , Especificidad por SustratoRESUMEN
We have synthesized an alternate substrate for trihydroxynaphthalene reductase (3HNR) and scytalone dehydratase (SD), two enzymes in the fungal melanin biosynthetic pathway. The oxidation of 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with concomitant reduction of NADP+ is catalyzed by 3HNR. DDBO is dehydrated by SD to 5-hydroxy-4H-1-benzopyran-4-one (HBO). These reactions can be monitored using continuous spectrophotometric assays. DDBO race-mizes rapidly, so chiral synthesis to mimic the natural substrate is not required. DDBO, DBO, and HBO are stable in aerated aqueous solution, in contrast to the rapidly autooxidizing trihydroxynaphthalene, a physiological substrate for 3HNR and product of SD. Unlike the natural substrates, DDBO, DBO, and HBO do not change protonation state between pH's 4 and 9. Oxidation of DDBO is effectively irreversible at pH 7, as DBO deprotonates with a pKa of 2.5. At pH 7.0 and 25 degrees C, the kcat for 3HNR catalyzed DDBO oxidation is 14 s-1 and the K(m) is 5 microM; the kcat for SD catalyzed DDBO dehydration is 400 s-1 and the K(m) is 15 microM. Based on these kinetic constants, DDBO is a better substrate than the natural substrate scytalone for both 3HNR and SD at neutral pH. An explanation for the preference of DDBO over scytalone in the oxidation and dehydration reactions is offered.
Asunto(s)
Benzopiranos/metabolismo , Proteínas Fúngicas , Hongos/metabolismo , Melaninas/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Ascomicetos/metabolismo , Benzopiranos/síntesis química , Hidroliasas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Oxidorreductasas/metabolismo , Espectrofotometría , Especificidad por SustratoRESUMEN
Scytalone dehydratase (SD) is a molecular target of inhibitor design efforts aimed at protecting rice plants from the fungal disease caused by Magnaporthe grisea. As determined from X-ray diffraction data of an SD-inhibitor complex [Lundqvist et al. (1994) Structure (London) 2, 937-944], there is an extended hydrogen-bonding network between protein side chains, the inhibitor, and two bound water molecules. From models of SD complexed to quinazoline and benztriazine inhibitors, a new class of potent SD inhibitors involving the displacement of an active-site water molecule were designed. We were able to increase inhibitory potency by synthesizing compounds with a nitrile functionality displayed into the space occupied by one of the crystallographic water molecules. Sixteen inhibitors are compared. The net conversion of potent quinazoline and benztriazine inhibitors to cyanoquinolines and cyanocinnolines increased binding potency 2-20-fold. Replacement of the nitrile with a hydrogen atom lowered binding affinity 100-30,000-fold. X-ray crystallographic data at 1.65 A resolution on a SD-inhibitor complex confirmed that the nitrile functionality displaced the water molecule as intended and that a favorable orientation was created with tyrosines 30 and 50 which had been part of the hydrogen-bonding network with the water molecule. Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency: one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Hidroliasas/antagonistas & inhibidores , Agua/química , Aminoquinolinas/síntesis química , Asparagina/metabolismo , Sitios de Unión/efectos de los fármacos , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Hidroliasas/química , Hidroliasas/metabolismo , Sustancias Macromoleculares , Magnaporthe/enzimología , Modelos Moleculares , Piridazinas/síntesis química , Quinazolinas/síntesis química , Salicilamidas/síntesis química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Ascomicetos/enzimología , Proteínas Fúngicas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Tiazoles/farmacología , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Simulación por Computador , Concentración de Iones de Hidrógeno , Cinética , NADP/química , Naftoles/química , Oxidorreductasas/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
BACKGROUND: Rice blast is caused by the pathogenic fungus,-Magnaporthe grisea. Non-pathogenic mutants have been identified that lack enzymes in the biosynthetic pathway of dihydroxynapthalene-derived melanin. These enzymes are therefore prime targets for fungicides designed to control rice blast disease. One of the enzymes identified by genetic analysis as a disease determinant is scytalone dehydratase. RESULTS: The three-dimensional structure of scytalone dehydratase in complex with a competitive inhibitor has been determined at 2.9 A resolution. A novel fold, a cone-shaped alpha + beta barrel, is adopted by the monomer in this trimeric protein, burying the hydrophobic active site in its interior. The interactions of the inhibitor with the protein side chains have been identified. The similarity of the inhibitor to the substrate and the side chains involved in binding afford some insights into possible catalytic mechanisms. CONCLUSIONS: These results provide a first look into the structure and catalytic residues of a non-metal dehydratase, a large class of hitherto structurally uncharacterized enzymes. It is envisaged that a detailed structural description of scytalone dehydratase will assist in the design of new inhibitors for controlling rice blast disease.