EVIDENCE FOR THE INVOLVEMENT OF 3-OXO-DELTA(4) INTERMEDIATES IN ECDYSTEROID BIOSYNTHESIS

Although the involvement of 3-oxo-Delta(4) compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long t ime, it has not yet been directly demonstrated. In the present study, 3-oxo-Delta(4)-steroids have been synthesized and incubated in vitro w ith dissociated moulting gland cells from the crab Carcinus maenas. Th e tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-Delta(4) compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-o xo-Delta(4)-steroid 4,7-cholstadien-14 alpha-ol-3,6-dione may be an in termediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-Delta(4) substrates. This reaction was the m ost active in crab Y-organs, as compared with other tissues. The chara cteristics of the 5 beta-reductase (subcellular localization, substrat e and cofactor requirements) appear similar to those of the vertebrate 3-oxo-Delta(4)-steroid 5 beta-reductase involved in steroid hormone c atabolism and bile acid biosynthesis.