Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3α-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis

Abstract

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3α-reductase to 3-epiecdysone. The 3DE 3α-reductase has been purified and subjected to limited amino acid sequencing. It occurs as two distinct forms, including a probable trimer of subunit molecular mass of approx. 26 kDa. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.2 kb) encoding the 26 kDa protein. Northern blotting showed that the mRNA transcript was expressed in Malpighian tubules during the early stage of the last larval instar. Conceptual translation of the 3DE 3α-reductase cDNA and database searching revealed that the enzyme belongs to the short-chain dehydrogenases/reductases superfamily. Furthermore, the enzyme is a novel eukaryotic 3-dehydrosteroid 3α-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3α-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3α-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3α-reductase and a previously reported 3DE 3β-reductase, which acts on the same substrate and belongs to the AKR family.