Molecular Cloning, Overexpression, and Characterization of Steroid-inducible 3a-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni A NOVEL MEMBER OF THE SHORT-CHAIN DEHYDROGENASE/REDUCTASE SUPERFAMILY*

3a-Hydroxysteroid dehydrogenase/carbonyl reductase (3a-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 3a-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 Nterminal sequence of the purified enzyme. The 3aHSD/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 3a-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 3aHSD/CR sequence) essential for catalytic activity of SDR proteins. 3a-HSD/CR was overexpressed and purified to homogeneity, and its activity was determined for steroid and nonsteroidal carbonyl substrates. These results suggest that inducible 3a-HSD/CR from C. testosteroni is a novel member of the SDR superfamily.