3 α-Hydroxysteroid-5β-oxidoreductase in Tissue Cultures of Digitalis lanata

Abstract

Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5β-pregnane-3,20-dione or 5β-pregnan-3β-ol-20-one, were administered to light- or darkgrown shoot cultures of Digitalis lanata. The unsaturated com pounds were reduced to their respective 5 a-pregnanes, 5β-pregnane-3,20-dione was reduced to 5β-pregnan-3α-ol-20-one and 5β-pregnan-3β-ol-20-one was isomerized to the respective 3α-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3α- and the 3β-isom er of 5β-pregnan-3-ol-20-one when incubated in the presence of 5β-pregnane- 3.20-dione. When 5β-pregnan-3α-ol-20-one was administered the cultured cells accumulated large amounts of the 3β-isomer together with small amounts of 5β-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 5β-pregnane- 3.20-dione almost exclusively to 5β-pregnan-3α-ol-20-one when 0.05 m MgCl2 were present in the incubation mixture. Under these conditions the formation of 5β-pregnan-3β-ol-20-one was inhibited. The enzyme activity could be recovered from m em brane-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 NADPH -dependent. SH reagents were essential for enzyme activity. The enzyme seems to be specific for 5β-pregnan-3-ones since neither 5 a-pregnane-3-ones nor Δ4/Δ5-pregnenes were reduced. The NADPH : 5β-pregnane 3α-hydroxysteroid-5β-oxidoreductase described here may play a role in the regulation of cardenolide biosynthesis by removing precursors, such as 5β-pregnane-3,20-dione, from the pathway

The biosynthesis of steroids and triterpenoids

“…1218,1221,1222 (N.B. progesterone 5 -reductase 1220 and 3 -hydroxysteroid 5 -oxidoreductase 1223 are also normally present and may serve to remove precursors such as 35 and 114 from the cardenolide pathway, thereby contributing to the overall regulation of cardenolide biosynthesis). Formation of the butenolide ring (117) has been suggested to involve transfer of a malonyl moiety to the 21-hydroxy group to form a malonyl hemi-ester (116) (catalysed by malonyl-coenzyme A: 21-hydroxypregnane 21-hydroxy-malonyltransferase) and subsequent Claisen-type condensation of the malonyl moiety with the C-20 ketone, accompanied by decarboxylation 1224 (rather than Claisen-type condensation as the initial step, as previously assumed).…”

Effects of Various Pregnanes and Two 23-Nor-5-cholenic Acids on Cardenolide Accumulation in Cell and Organ Cultures ofDigitalis lanata

“…(Scrophulariaceae) were propagated in MS medium (9) without phytohormones. The suspensions were grown in 1-I shake flasks kept in the dark on gyratory shakers; they were maintained and subcultivated as described byKreis and Reinhard (10),, Shoot cultures of D. lanata were initiated from axillary buds and maintained in 300-mI shake flasks in 50 ml liquid medium as described (8,11).…”

“…Cardenolide synthesis is restored when dark-grown cultures are exposed to light. The major cardenolides accumulating in lightgrown shoots have been identified and quantified recently (8). The main objective of the experiments described here was to examine the effects of various pregnane precursors on the accumulation of different types of cardenolides.…”