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J. Biol. Chem., Vol. 276, Issue 29, 26819-26828, July 20, 2001

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Regulation of Ecdysteroid Signaling: Cloning and Characterization of Ecdysone Oxidase
A NOVEL STEROID OXIDASE FROM THE COTTON LEAFWORM, SPODOPTERA LITTORALIS^*

Hajime Takeuchi, Jian-Hua Chen, David R. O'Reilly, Philip C. Turner, and Huw H. Rees^§

From the Cellular Regulation and Signaling Division, School of Biological Sciences, University of Liverpool, Life Sciences Bldg., Crown Street, Liverpool, L69 7ZB, and  Department of Biology, Sir Alexander Fleming Bldg., Imperial College of Science, Technology and Medicine, Imperial College Road, South Kensington, London, SW7 2AZ, United Kingdom

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 single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AY035784 and AY035785.

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