The catalytic mechanism of the 3-ketosteroid isomerase of Digitalis lanata involves an intramolecular proton transfer and the activity is not associated with the 3β-hydroxysteroid dehydrogenase activity

•Reaction mechanism of Digitalis lanata 3KSI is investigated.•Isoprogesterone isomerization proceeds via an intramolecular proton transfer.•The mechanism is consistent with the mechanism of the bacterial 3KSIs.•rDl3βHSD does not possess 3-ketosteroid isomerase activity.