3 a-Hydroxysteroid-5 ß-oxidoreductase in Tissue Cultures of Digitalis lanata

U rsel S tuh lem m er, W ern er H aussm ann , F ran k M ilek , W olfgang K reis, and E rn s t R e in h a rd Pharmazeutisches Institut, Eberhard-Karls-Universität, A uf der Morgenstelle 8 D-72076 Tübingen, Bundesrepublik Deutschland Z. Naturforsch. 48 c, 713-721 (1993); received July 23,1993 Digitalis lanata, Biotransformation, Cardenolide Biosynthesis, Cell-Free System, 3 a-Hydroxysteroid-5 ß-oxidoreductase Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5 ß-pregnane-3,20-dione or 5 ß-pregnan-3 ß-ol-20-one, were administered to lightor darkgrown shoot cultures of Digitalis lanata. The unsaturated compounds were reduced to their respective 5 a-pregnanes, 5 ß-pregnane-3,20-dione was reduced to 5 ß-pregnan-3 a-ol-20-one and 5 ß-pregnan-3 ß-ol-20-one was isomerized to the respective 3 a-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3 aand the 3ß-isomer of 5ß-pregnan-3-ol-20-one when incubated in the presence of 5ß-pregnane3.20-dione. When 5 ß-pregnan-3 a-ol-20-one was administered the cultured cells accumulated large amounts of the 3 ß-isomer together with small amounts of 5 ß-pregnane-3,20-dione, which may be regarded as an intermediate during the isomerization reaction. Cell-free, buffered extracts from light-grown shoots were shown to reduce 5 ß-pregnane3.20-dione almost exclusively to 5 ß-pregnan-3 a-ol-20-one when 0.05 m MgCl2 were present in the incubation mixture. U nder these conditions the formation of 5 ß-pregnan-3 ß-ol-20-one was inhibited. The enzyme activity could be recovered from membrane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 °C. The energy of activation was 56.2 kJ/mol and the enzyme reaction was found to be N ADPH-dependent. SH reagents were essential for enzyme activity. The enzyme seems to be specific for 5 ß-pregnan-3-ones since neither 5 a-pregnane-3-ones nor A4/A5-pregnenes were reduced. The N A D P H : 5 ß-pregnane 3 a-hydroxysteroid-5 ß-oxidoreductase described here may play a role in the regulation of cardenolide biosynthesis by removing precursors, such as 5 ß-pregnane-3,20-dione, from the pathway.