Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

Abstract

3β-Hydroxysteroid oxidase (3β-hydroxysteroid: oxygen oxidoreductase, EC 1.1.3.6.) from the culture supernatant of Brevibacterium sterolicum ATCC 21387 has a molecular weight of 32,500 and an isoelectric point of 8.9. The enzyme contained 258 amino acid residues and the composition revealed a distinctive feature of a relatively high amount of proline and the absence of alanine and tryptophan. The crystalline enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 280, 390, and 470 nm with a shoulder at 490 nm. Anaerobic addition of dehydro-epi-androsterone as well as sodium dithionite to the enzyme produced a disappearance of the peaks at 390 and 470 nm. The flavin moiety of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of protein. The enzyme is sulfhydryl dependent and was inactivated by silver and mercury compounds. Analysis of the enzyme protein by atomic absorption spectrophotometry failed to detect any significant quantity of heavy metals. Various 3β-hydroxysteroids were oxidized and the relative rates of the oxidation were cholesterol, 100; dehydro-epi-androsterone, 41; pregnenolone, 22; and β-sitosterol, 20. The oxidation product of cholesterol by the enzyme was crystallized and identified as 4-cholesten-3-one by melting point, elementary analysis, optical rotation, UV, IR and NMR spectra. The oxidation of cholesterol proceeded as follows: The enzyme would be used for some analytical and preparative purposes in the field of steroid chemistry, e.g., microdetermination of cholesterol in serum.