Δ5-3β-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 fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column 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 digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3 beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.