The inducible 3-keto-5-alpha-steroid-DELTA-4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-northestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and special analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of M(r) 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-northestosterone. 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.
