RESUMO
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.
Assuntos
Aeropyrum/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Aeropyrum/genética , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Animais , Proteínas Arqueais/genética , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Domínio Catalítico , Galinhas , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Genes Arqueais , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
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.
Assuntos
Aldeído Redutase/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/enzimologia , Regulação Enzimológica da Expressão Gênica , Hipotireoidismo/fisiopatologia , Fígado/enzimologia , Aldeído Redutase/química , Aldeído Redutase/genética , Aldo-Ceto Redutases , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Biocatálise , Domínio Catalítico , Galinhas , Bases de Dados de Proteínas , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Fenantrenos/metabolismo , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina/químicaRESUMO
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%.
