RESUMEN
Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene.
Asunto(s)
Inhibidores Enzimáticos/química , Oxidorreductasas/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , ADN Complementario , Dimerización , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Sondas Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Estructura Secundaria de ProteínaRESUMEN
Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21-NADPH binary complex was determined from an orthorhombic crystal belonging to space group P2(1)2(1)2(1) at a resolution of 1.8 A. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3alpha-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme-coenzyme-substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site.
Asunto(s)
Holoenzimas/química , Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/fisiología , Sitios de Unión/fisiología , Cristalización , Holoenzimas/fisiología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/fisiología , Especificidad por Sustrato/fisiologíaRESUMEN
20alpha-hydroxysteroid dehydrogenase (AKR1C1) plays a key role in the metabolism of progesterone and other steroid hormones, thereby regulating their action at the pre-receptor level. AKR1C1 is implicated in neurological and psychiatric conditions such as catamenial epilepsy and depressive disorders. Increased activity of AKR1C1 is associated with termination of pregnancy and the development of breast cancer, endometriosis and endometrial cancer. Inhibition of the undesired activity of AKR1C1 will help reduce risks of premature birth, neurological disorders and the development of cancer. In order to identify potential leads for new inhibitors of AKR1C1 we adopted a virtual screening-based approach using the automated DOCK program. Approximately 250,000 compounds from the NCI database were screened for potential ligands based on their chemical complementarity and steric fit within the active site of AKR1C1. Kinetic analysis revealed 3,5-diiodosalicylic acid, an analogue of salicylic acid, as a potent competitive inhibitor with respect to the substrate 5beta-pregnane-3alpha,20alpha-diol with a K(i) of 9 nM. Aspirin, which is a well known salicylic acid-based drug, was also found to inhibit AKR1C1 activity. This is the first report to show aspirin (IC(50)=21 microM) and its metabolite salicylic acid (IC(50)=7.8 microM) as inhibitors of AKR1C1.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Ácido Salicílico/química , Sitios de Unión , Bases de Datos Factuales , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Ligandos , Unión ProteicaRESUMEN
Structure determination of porcine aldehyde reductase holoenzyme in complex with the potent aldose reductase inhibitor fidarestat was carried out to explain the difference in the potency of the inhibitor for aldose and aldehyde reductases. The hydrogen bonds between the active-site residues Tyr50, His113, and Trp114 and fidarestat are conserved in the two enzymes. In aldose reductase, Leu300 forms a hydrogen bond through its main-chain nitrogen atom with the exocyclic amide group of the inhibitor, which when replaced with a Pro in aldehyde reductase, cannot form a hydrogen bond, thus causing a loss in binding energy. Furthermore, in aldehyde reductase, the side chain of Trp220 occupies a disordered split conformation that is not observed in aldose reductase. Molecular modeling and inhibitory activity measurements suggest that the difference in the interaction between the side chain of Trp220 and fidarestat may contribute to the difference in the binding of the inhibitor to the enzymes.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/química , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/química , Imidazolidinas/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Holoenzimas/química , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , PorcinosRESUMEN
Aldose reductase, a monomeric NADPH-dependent oxidoreductase, catalyzes the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The X-ray structure of human aldose reductase holoenzyme in complex with statil was determined at a resolution of 2.1 A. The carboxylate group of statil interacted with the conserved anion binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Statil's hydrophobic phthalazinyl ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group penetrating the "specificity" pocket. The interactions between the inhibitor's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding to residues Leu300 and Thr113. Based on the model of the ternary complex, the program GRID was used in an attempt to design novel potential inhibitors of human aldose reductase with enhanced binding energies of the complex. Molecular modeling calculations suggested that the replacement of the fluorine atom of statil with a carboxylate functional group may enhance the binding energies of the complex by 33%.
Asunto(s)
Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Ftalazinas/química , Ftalazinas/metabolismo , Aldehído Reductasa/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
L-Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 A resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N-terminal residues of subunits related by the P-axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L-xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased K(m) for the substrate (>26-fold) and K(d) for NADPH (95-fold), but only the N107L mutation significantly decreased k(cat) value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism.
Asunto(s)
Asparagina/química , Modelos Moleculares , Deshidrogenasas del Alcohol de Azúcar/química , Secuencia de Aminoácidos , Asparagina/genética , Asparagina/fisiología , Sitios de Unión , Cristalografía por Rayos X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
Dihydrodiol dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric dihydrodiol dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.
Asunto(s)
Oxidorreductasas/química , Animales , Cristalización , Cristalografía por Rayos X , Dimerización , Macaca , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Human L-xylulose reductase was crystallized from buffered polyethylene glycol solutions using the hanging-drop vapour-diffusion method. The crystals diffract to 2.1 A resolution and belong to the orthorhombic P222 space group, with unit-cell parameters a = 72.9, b = 74.1, c = 87.9 A. This is the first crystallization report of a xylulose reductase that is identical to diacetyl reductase.
Asunto(s)
Deshidrogenasas del Alcohol de Azúcar/química , Cristalización , Cristalografía por Rayos X , Humanos , Estructura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificaciónRESUMEN
Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography. Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method. X-ray data were collected to 2.75 A resolution. The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees. This is the first crystallization report of human sorbitol dehydrogenase.