Cloning, expression and characterization of a novel short-chain dehydrogenase/reductase (SDRx) in Comamonas testosteroni.

The short-chain dehydrogenase/reductase (SDR) superfamily is a large and diverse group of genes with members found in all forms of life. Comamonas testosteroni ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In previous investigations, we have identified 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from C. testosteroni as a member of the SDR superfamily that catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus of a great variety of C(19-27) steroids. In addition, 3α-HSD/CR was shown to mediate the carbonyl reduction of non-steroidal aldehydes and ketones. Interestingly, the 3α-HSD/CR gene (hsdA) expression is induced by steroids such as testosterone and progesterone. In the present investigation, we found a novel SDR gene (SDRx) which is located 3.6kb downstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this SDRx consists of 768bp and translates into a protein of 255 amino acids. 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 160-164 in the SDRx sequence) essential for catalytic activity of SDR proteins. Phylogenetic analyses indicated that the novel SDRx gene codes for 7α-hydroxysteroid dehydrogenase (7α-HSD) in C. testosteroni which is active in steroid metabolism. To produce purified SDRx protein, the SDRx gene was cloned into plasmid pET-15b and the overexpressed protein was purified by its His-tag sequence on metal chelate chromatography. To prove that SDRx is involved in the metabolic pathway of steroid compounds, we constructed an SDRx knock-out mutant of C. testosteroni. Compared to wild type C. testosteroni, degradation of the steroids testosterone and estradiol decreased in the SDRx knock-out mutant. Furthermore, growth on the steroids cholic acid, estradiol and testosterone was impaired in the SDRx knock-out strain. Combined, the novel SDRx in C. testosteroni was identified as 7α-HSD that is involved in steroid degradation. Article from a special issue on steroids and microorganisms.