title: Purification of Δ(5)-3-ketosteroid isomerase from Digitalis lanata.
abstract: The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ(5)-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ(5)-3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS-PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.
authors.affiliation: Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany. Electronic address: Nadine.meitinger@fau.de.
authors.forename: Nadine
authors.initials: N
authors.lastname: Meitinger
authors.affiliation: Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Geiger
authors.affiliation: Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany.
authors.forename: Thierry W
authors.initials: TW
authors.lastname: Augusto
authors.affiliation: Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany; Faculdade de Farmácia, Universidade Federal de Minas Gerais (UFMG), Av. Antônio Carlos, 6627, 31270-010 Belo Horizonte, Brazil.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Maia de Pádua
authors.affiliation: Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kreis
mesh_terms: D004070:Digitalis / Q000201:enzymology*; D015394:Molecular Structure; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000302:isolation & purification*; D011374:Progesterone / Q000031:analogs & derivatives / Q000737:chemistry; D013251:Steroid Isomerases / Q000302:isolation & purification*
chemical_list: C485330:4-pregnene-3,20-dione; D010940:Plant Proteins; D011374:Progesterone; D013251:Steroid Isomerases; C022660:steroid delta-isomerase
keywords: Cardenolides; Digitalis lanata; Isoprogesterone; Progesterone; Δ(5)-3-Ketosteroid isomerase