Evidence for the involvement of 3-oxo-Δ4 intermediates in ecdysteroid biosynthesis

Abstract

Although the involvement of 3-oxo-delta 4 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-delta 4-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-delta 4 compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-delta 4-steroid 4,7-cholestadien-14 alpha-ol-3,6-dione may be an intermediate 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 most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-delta 4-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

“…An alternative possibility is that Nm-g/Sro might act as an ecdysteroid 5-reductase converting  4 -diketol to diketol. This is the putative last step in the Black Box proposed from studies using the Y-organ of the crab Carcinus maenas (Blais et al, 1996), although  4 -diketol has neither been isolated nor shown to be a product in the ecdysteroid biosynthesis pathway in insects . Further studies to identify the exact step in which Nmg/Sro is involved would shed light on the molecular mechanisms controlling developmental timing in insects.…”

Steroid signaling in plants and insects—common themes, different pathways

“…Surprisingly, however, it should be noted that there is no good evidence for a 5␣-reductase activity in insects. 5␣-compounds have no biological activity in arthropods and have not been detected among the secretory products from molting glands (Bergamasco and Horn 1980;Blais et al 1996). This suggests that the genes encoding steroid 5␣-reductases have been lost in the insect lineage.…”

Crustacean Molting: Regulation and Effects of Environmental Toxicants

“…An inhibitor of steroid 5-reductase, L-645390, blocks the conversion of cholesterol to 7DC in the YO of M. mercenaria [18]. The 5-reductase that converts 4-diketol to 5-diketol is a cytosolic enzyme in YO cells that requires NADPH for activity [19]. Orthologs of nvd, nmg/sro, spo, phm, dib, sad, and shd have been identified in the Daphnia pulex genome [20][21][22], and a cDNA encoding Phm has been cloned from Kuruma prawn, Marsupenaeus japonicus [23].…”