Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase 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 (C. testosterone) ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In the present investigation, we found a novel SDR gene 7alpha-hydroxysteroid dehydrogenase (7α-HSD) which is located 11.9 kb upstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this putative 7alpha-hydroxysteroid dehydrogenase gene consists of 771 bp and translates into a protein of 256 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 161-165 in the 7α-HSD sequence) essential for catalytic activity of SDR proteins. To produce purified 7α-HSD protein, the 7α-HSD gene was cloned into plasmid p(ET-15b) and the over expressed protein was purified by His-tag sequence on metal chelate chromatography. To prove that 7α-HSD is involved in the metabolic pathway of steroid compounds, we constructed a 7α-HSD knock-out mutant of C. testosteroni. Compared to the wild type C. testosteroni, degradation of testosterone, estradiol and cholesterol were decreased in the 7α-HSD knock-out mutant. Furthermore, growth in the medium with testosterone, estradiol and cholesterol was impaired in 7α-HSD knock-out mutant. The results showed that 7α-HSD is involved in steroid degradation.
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