Molecular cloning, overexpression, and characterization of steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short-chain dehydrogenase/reductase superfamily.
3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a bacterium that is able to grow on steroids as the sole carbon source, catalyzes the oxidoreduction at position 3 of a variety of C19-27 steroids and the carbonyl reduction of a variety of nonsteroidal aldehydes and ketones. The gene of this steroid-inducible 3alpha-HSD/CR was cloned by screening a C. testosteroni gene bank with a homologous DNA probe that was obtained by polymerase chain reaction with two degenerative primers based on the N-terminal sequence of the purified enzyme. The 3alpha-HSD/CR gene is 774 base pairs long, and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed that amino acid sequences highly conserved in the short-chain dehydrogenase/reductase (SDR) superfamily are present in 3alpha-HSD/CR. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 155-159 in the 3alpha-HSD/CR sequence) essential for catalytic activity of SDR proteins. 3alpha-HSD/CR was overexpressed and purified to homogeneity, and its activity was determined for steroid and nonsteroidal carbonyl substrates. These results suggest that inducible 3alpha-HSD/CR from C. testosteroni is a novel member of the SDR superfamily.
Möbus
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Department of Pharmacology and Toxicology, School of Medicine, Philipps University of Marburg, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany.
Maser
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D015096:3-Hydroxysteroid Dehydrogenases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004790:Enzyme Induction; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000494:pharmacology*
D011994:Recombinant Proteins; D013256:Steroids; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)