Regulation of Ecdysteroid Signaling: Cloning and Characterization of Ecdysone Oxidase

Abstract

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3␣-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein.Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) ؉ -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a singlecopy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori

“…However, previous studies did not show the enzyme activity of the ecdysone oxidase. In addition, the putative silkworm ecdysone oxidase gene was found to be mainly expressed in the hemocytes 22, which was different from the other studies (Midgut is the predominantly expressed tissue for the EO gene from both S. littoralis and D. melanogaster ) 13-14. Therefore, whether the gene is the homologue of the ecdysone oxidase gene in the silkworm or not is still unknown.…”

“…Then the genes which encode the two enzymes have been cloned and characterized in several insects. For example, the genes encoding EO and 3DE-3α-reductase in S. littoralis have been cloned and characterized in previous studies 12-13. Moreover, EO was also identified in Drosophila melanogaster by the same researchers 14.…”

Biosynthesis and potential functions of the ecdysteroid 20-hydroxyecdysone — a review

“…E and 20E may be transformed, respectively, into 26-hydroxyecdysone (26E) and 20,26-hydroxyecdysone (20,26E) by an unidentified P450 enzyme (Warren et al 2006). In Drosophila melanogaster and Spodoptera littoralis, E may be oxidized by ecdysone oxidase, producing 3-dehydroecdysone (Takeuchi et al 2001;Takeuchi et al 2005). In S. littoralis, 3-dehydroecdysone may be irreversibly reduced to 3-epiecdysone by 3-dehydroecdysone (3DE) 3a-reductase (Takeuchi et al 2000).…”

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

“…Previous studies reported high EO activity during the early pupal stage in many insects although the functional significance is unknown [13]. We investigated spatial and temporal expression patterns of BmEO in three development stages and 10 tissues using qRT-PCR.…”

“…20E conversion to 3-dehydroecdysone (3DE) can be catalysed by ecdysone oxidase (EO), which could be reversed by 3-dehydroecdysone 3b-reductase [13]. The intermediate, 3DE, is converted to 3-epiecdysone by 3-dehydroecdysone 3a-reductase irreversibly [13,14]. As one of the key enzymes in this pathway, EO was first identified and purified from the dipteran insect Calliphora vicina.…”