Enzyme Activities in Cardenolide-Accumulating, Mixotrophic Shoot Cultures of Digitalis purpurea L.

Abstract

Summary Photomixotrophic, aseptically growing shoot cultures of Digitalis purpurea accumulate cardiac glycosides. All cardenolides are 5β-configurated. The progesterone 5β-reductase and the 3β-hydroxysteroid-5β-oxidoreductase present in these cultures are involved in providing precursors for cardenolide synthesis, whereas a progesterone 5α-reductase also present in shoot cultures is discussed with regard to its role in phytosterol biosynthesis. The 3β-hydroxysteroid-5β-oxidoreductase catalyzed the reduction of 5β-pregnane-3,20-dione to 5β-pregnan-3β-ol-20-one, which is a precursor of cardenolides; the reaction was reversible. The progesterone 5α-reductase had an activity maximum 7 days after the onset of cultivation whereas progesterone 5β-reductase had its maximum on day 11. The cardenolide content of the plantlets was highest after 24 days. The soluble 30-hydroxysteroid-5β-oxidoreductase had a pH-optimum at 6.5; the temperature optimum was 40 °C. The apparent K m value for the substrate 5β-pregnane-3,20-dione was 17.1 μM. The enzyme utilizes NADPH 2 and NADH 2 as co-substrates with an apparent K m -value for NADPH 2 of 0.8 μM.