Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3 alpha-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis.
One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3 alpha-reductase to 3-epiecdysone. The 3DE 3 alpha-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 3 alpha-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 3 alpha-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3 alpha-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3 alpha-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3 alpha-reductase and a previously reported 3DE 3 beta-reductase, which acts on the same substrate and belongs to the AKR family.
Takeuchi
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H
School of Biological Sciences, University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, U.K.
Chen
J H
JH
O'Reilly
D R
DR
Rees
H H
HH
Turner
P C
PC
D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D026461:Ecdysteroids; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018411:Spodoptera / Q000201:enzymology*; D013256:Steroids / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution
D018076:DNA, Complementary; D026461:Ecdysteroids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013256:Steroids; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; C104053:3-dehydroecdysone 3alpha-reductase