RESUMEN
A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10min up to 90°C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher Km values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme's lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR.
Asunto(s)
Aeropyrum/enzimología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Aeropyrum/genética , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Proteínas Aviares/química , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Dominio Catalítico , Pollos , Clonación Molecular , Cristalografía por Rayos X , Estabilidad de Enzimas , Genes Arqueales , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
A gene encoding a functionally unknown protein that is specifically expressed in the thyroidectomized chicken fatty liver and has a predicted amino acid sequence similar to that of NAD(P)H-dependent carbonyl reductase was overexpressed in Escherichia coli; its product was purified and characterized. The expressed enzyme was an NAD(P)H-dependent broad substrate specificity carbonyl reductase and was inhibited by arachidonic acid at 1.5 µm. Enzymological characterization indicated that the enzyme could be classified as a cytosolic-type carbonyl reductase. The enzyme's 3D structure was determined using the molecular replacement method at 1.98 Å resolution in the presence of NADPH and ethylene glycol. The asymmetric unit consisted of two subunits, and a noncrystallographic twofold axis generated the functional dimer. The structures of the subunits, A and B, differed from each other. In subunit A, the active site contained an ethylene glycol molecule absent in subunit B. Consequently, Tyr172 in subunit A rotated by 103.7° in comparison with subunit B, which leads to active site closure in subunit A. In Y172A mutant, the Km value for 9,10-phenanthrenequinone (model substrate) was 12.5 times higher than that for the wild-type enzyme, indicating that Tyr172 plays a key role in substrate binding in this carbonyl reductase. Because the Tyr172-containing active site lid structure (Ile164-Gln174) is not conserved in all known carbonyl reductases, our results provide new insights into substrate binding of carbonyl reductase. The catalytic properties and crystal structure revealed that thyroidectomized chicken fatty liver carbonyl reductase is a novel enzyme.
Asunto(s)
Aldehído Reductasa/metabolismo , Modelos Animales de Enfermedad , Hígado Graso/enzimología , Regulación Enzimológica de la Expresión Génica , Hipotiroidismo/fisiopatología , Hígado/enzimología , Aldehído Reductasa/química , Aldehído Reductasa/genética , Aldo-Ceto Reductasas , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Biocatálisis , Dominio Catalítico , Pollos , Bases de Datos de Proteínas , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Fenantrenos/metabolismo , Conformación Proteica , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina/químicaRESUMEN
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=104.26, b=81.32, c=77.27â Å, ß=119.43°, and diffracted to 1.86â Å resolution on beamline NE3A at the Photon Factory. The overall Rmerge was 5.4% and the data completeness was 99.4%.