Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase in Comarnonas 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 (7 alpha-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 GlyX-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 161-165 in the 7 alpha-HSD sequence) essential for catalytic activity of SDR proteins. To produce purified 7 alpha-HSD protein, the 7 alpha-HSD gene was cloned into plasmid pET-151' and the over expressed protein was purified by His-tag sequence on metal chelate chromatography. To prove that 7 alpha-HSD is involved in the metabolic pathway of steroid compounds, we constructed a 7 alpha-HSD knock-out mutant of C testosteroni. Compared to the wild type C testosteroni, degradation of testosterone, estradiol and cholesterol were decreased in the 7 alpha-HSD knockout mutant. Furthermore, growth in the medium with testosterone, estradiol and cholesterol was impaired in 7aHSD knock-out mutant. The results showed that 7 alpha-HSD is involved in steroid degradation. (C) 2013 Elsevier GmbH. All rights reserved.