title: Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase in Comamonas testosteroni.
abstract: 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.
authors: Jilin University, College of Animal Science and Veterinary Medicine, Changchun, PR China; Changchun University of Science and Technology, School of Life Science and Technology, Changchun, PR China.
authors: Wei
authors: W
authors: Ji
authors: Changchun University of Science and Technology, School of Life Science and Technology, Changchun, PR China.
authors: Yuanan
authors: Y
authors: Chen
authors: Changchun University of Science and Technology, School of Life Science and Technology, Changchun, PR China.
authors: Hao
authors: H
authors: Zhang
authors: Changchun University of Science and Technology, School of Life Science and Technology, Changchun, PR China.
authors: Xiao
authors: X
authors: Zhang
authors: Jilin University, College of Animal Science and Veterinary Medicine, Changchun, PR China. Electronic address: ziyi@jlu.edu.cn.
authors: Ziyi
authors: Z
authors: Li
authors: Changchun University of Science and Technology, School of Life Science and Technology, Changchun, PR China. Electronic address: yuyuanhua8888@126.com.
authors: Yuanhua
authors: Y
authors: Yu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase
keywords: 7α-HSD; 7α-HSD knock-out mutant; Comamonas testosteroni; Short-chain dehydrogenase/reductase