Spectral properties of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina.
3-Ketosteroid-delta 1-dehydrogenase from Nocardia corallina is a flavoenzyme that catalyzes 1,2-desaturation of 3-ketosteroid. The dehydrogenase generated complexes with 3-ketosteroids and phenolic steroids such as estradiol with remarkable perturbations of the visible spectrum. The enzyme did not make the adduct with sulfite ion, but could use molecular oxygen as the electron acceptor. The CD spectra of oxidized and steroid-bound enzymes exhibited positive dichroisms in the visible region which resembled those of flavoenzyme oxidases. The dehydrogenase led isosbestically to the stable red semiquinone species with large yields upon photochemical or dithionite reduction (at pH 7.4) in the presence of the steroid product, 1,4-androstadiene-3,17-dione, but in the absence of the steroid the yield of semiquinone was low and the fully reduced enzyme was obtained. Substrate titration also yielded the red flavo-semiquinone stoichiometrically and it was hard to generate the fully reduced form. The reduced enzyme was oxidized with molecular oxygen, but did not oxidize with ferricyanide. An EPR study of these half-reduced forms confirmed the presence of the radical species with the g = 2.004 signal. The dehydrogenase was rapidly reduced with an excess amount of 3-ketosteroid at about 80% yield at pH 7.4 under anaerobic conditions and the reduced species was altered to the stable red semiquinone species. The rate of this reaction was t1/2 = 28 min at pH 7.4, 130 min at pH 9.0 and 34 min at pH 6.4, respectively. These results indicate that the semiquinone species does not act directly in turnover of the dehydrogenase reaction. The results were compared with the spectral properties of general acyl-CoA dehydrogenases and acyl-CoA oxidase toward the mechanism of C1,2-dehydrogenation.
Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa, Japan.
E
E
Itagaki
T
T
Hatta
T
T
Wakabayashi
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K
Suzuki
D000735:Androstenedione / Q000378:metabolism; D002942:Circular Dichroism; D004227:Dithionite / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D009615:Nocardia / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D000735:Androstenedione; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase