3-keto-5 alpha-steroid-delta 4-dehydrogenase from Nocardia corallina: purification and characterization.
The inducible 3-keto-5 alpha-steroid-delta 4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5 alpha-steroid at the 4- and 5-position, e.g. the conversion of 5 alpha-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5 alpha-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4 beta, 5 alpha-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5 alpha-steroids but not 3 beta-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5 alpha-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-delta 1-dehydrogenase.
Hatta
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T
Department of Chemistry, Faculty of Science, Kanazawa University.
Wakabayashi
T
T
Itagaki
E
E
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology*; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry; D013256:Steroids / Q000302:isolation & purification; D013379:Substrate Specificity
D005420:Flavoproteins; D013256:Steroids; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase