Molecular cloning overexpression, and characterization of steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni -: A novel member of the short-chain dehydrogenase/reductase superfamily

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