Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?
Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-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-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-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)-3beta-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.
Finsterbusch
A
A
Universität Halle, Institut für Pharmazeutische Biologie, Hoher Weg 8, D-06120 Halle, Germany.
Lindemann
P
P
Grimm
R
R
Eckerskorn
C
C
Luckner
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D004070:Digitalis / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013251:Steroid Isomerases / Q000737:chemistry; D013256:Steroids / Q000378:metabolism; D013379:Substrate Specificity
D013256:Steroids; D011374:Progesterone; D011284:Pregnenolone; D015096:3-Hydroxysteroid Dehydrogenases; C031954:delta(5)-3 beta-hydroxysteroid dehydrogenase; D013251:Steroid Isomerases; C022660:steroid delta-isomerase