Although the involvement of 3-oxo-Δ4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-Δ4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-Δ4 compounds had undergone a 5β-reduction, to give the 5β-conformation of ecdysteroids. Our results suggest that the 3-oxo-Δ4-steroid 4,7-cholestadien-14α-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5β-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-Δ4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5β-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-Δ4-steroid 5β-reductase involved in steroid hormone catabolism and bile acid biosynthesis.
Present address: Institute of Bioorganic Chemistry, Byelorussian Academy of Sciences, 220141 Zhodinskaya, 5/2 Minsk, Byelorussia.