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J. Biochem, 1988, Vol. 103, No. 6 1039-1044
© 1988 Japanese Biochemical Society

research-article

Purification and Characterization of 17ß-Hydroxysteroid Dehydrogenase from Cylindrocarpon radicicola1

Eiji Itagaki, Faculty of Science and Teturou Iwaya, Faculty of Science

Department of Chemistry,Kanazawa University Kanazawa, Ishikawa 920

An NAD+-linked 17ß-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58, 600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26, 000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17ß-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax =85 umol/min/mg; Km for the steroid=9.5 µM; Km for NAD+=198 µM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 µmol/min/mg; Km for the steroid = 24 µM; Km for NADH=6.8 µM at pH 7.0). In the purified enzyme preparation, no activity of 3{alpha}-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase, {Delta}5–3-ketosteroid-4, 5-isomerase, or steroid ring A-{Delta}-dehydrogenase was detected. Among several steroids tested, only 17ß-hydroxysteroids such as testosterone, estradiol-17ß, and 11ß-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17{alpha}-3H]testosterone.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


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