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Purification of Δ5-3-ketosteroid isomerase from Digitalis lanata

2014; Elsevier BV; Volume: 109; Linguagem: Inglês

10.1016/j.phytochem.2014.10.025

1873-3700

Nadine Meitinger, Daniel Geiger, Thierry Waltrich Augusto, Rodrigo Maia de Pádua, Wolfgang Kreis,

Biochemical Acid Research Studies

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 Δ5-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ5-3β-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 17 kDa in SDS–PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.