Delta(5)-3 beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. -a multifunctional enzyme in steroid metabolism?

Delta(5)-3 beta-Hydroxysteroid dehydrogenase (Delta(5)-3 beta-HSD; EC 1.1.1 .145), an enzyme converting pregn-5-ene-3 beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fra ction of suspension-cultured cells of Digitalis lanata L. strain VIII. Star ting with acetone dry powder the enzyme was purified in three steps using c olumn chromatography on Fractogel-TSI( DEAE, hydroxyapatite and Sephacryl G -200. Fractions with highest Delta(5)-3 beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ d igestion the resulting bands were sequenced N-terminally. The 29-kDa band y ielded three fragments with high sequence homology to members of the superf amily of short-chain dehydrogenases/reductases. High similarity was found t o microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3 beta-HSD. The purified enzyme was characterized with respec t to kinetic parameters, substrate specificity and localization. The functi on of the enzyme in steroid metabolism is discussed.