Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina.
The inducible 3-ketosteroid-delta 1-dehydrogenase of Nocardia corallina which catalyzes the introduction of a double bond into the position of carbon 1 and 2 of ring A of 3-ketosteroid has been obtained in four steps with a 50% yield and 360-fold purification. The enzyme is homogeneous as judged by SDS-gel electrophoresis and is a monomeric protein with a molecular weight of 60,500. The isoelectric point of the enzyme is about 3.1. The enzyme contains 1 mol of flavin adenine dinucleotide per mol of protein, and has a typical flavoprotein absorption spectrum with maxima of 458, 362 and 268 nm. The enzyme is very stable in the absence of added cofactors, and catalyzes the dehydrogenation of delta 4-3-ketosteroids in the presence of phenazine methosulfate, which acts as an excellent electron acceptor. Potassium ferricyanide and cytochrome c did not act as electron acceptors. The delta 1-dehydrogenation was also stimulated by molecular oxygen with stoichiometric production of hydrogen peroxide and delta 1,4-3-ketosteroid. The optimum pH is 10 for dehydrogenation using phenazine methosulfate, and is between 8.5 and 10 for the oxidase reaction. The enzyme oxidizes a wide variety of 3-ketosteroids, but not 3 beta-hydroxysteroids. 3-Ketosteroids having an 11 alpha- or 11 beta-hydroxyl group were oxidized at slow rates. The purified enzyme catalyzes efficiently aromatization of the A-ring of 19-nortestosterone and 19-norandrostenedione to produce estradiol and estrone. 19-Hydroxytestosterone, 19-hydroxyandrostenedion and 19-oxotestosterone were converted to the respective phenolic steroids with cleavage of the C10 side-chain. Activities of 3-ketosteroid-delta 4-dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase were not observed in the purified preparations. Properties of this novel flavoprotein enzyme are discussed.
Itagaki
E
E
Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa, Japan.
Wakabayashi
T
T
Hatta
T
T
D000596:Amino Acids / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013057:Spectrum Analysis; D013379:Substrate Specificity
D000596:Amino Acids; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase