Cloning, expression and characterization of a novel short-chain dehydrogenase/reductase (SDRx) in Comamonas testosteroni. 2012

Wenjie Gong, and Guangming Xiong, and Edmund Maser
Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Schleswig-Holstein, Campus Kiel, Kiel, Germany.

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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010802 Phylogeny The relationships of groups of organisms as reflected by their genetic makeup. Community Phylogenetics,Molecular Phylogenetics,Phylogenetic Analyses,Phylogenetic Analysis,Phylogenetic Clustering,Phylogenetic Comparative Analysis,Phylogenetic Comparative Methods,Phylogenetic Distance,Phylogenetic Generalized Least Squares,Phylogenetic Groups,Phylogenetic Incongruence,Phylogenetic Inference,Phylogenetic Networks,Phylogenetic Reconstruction,Phylogenetic Relatedness,Phylogenetic Relationships,Phylogenetic Signal,Phylogenetic Structure,Phylogenetic Tree,Phylogenetic Trees,Phylogenomics,Analyse, Phylogenetic,Analysis, Phylogenetic,Analysis, Phylogenetic Comparative,Clustering, Phylogenetic,Community Phylogenetic,Comparative Analysis, Phylogenetic,Comparative Method, Phylogenetic,Distance, Phylogenetic,Group, Phylogenetic,Incongruence, Phylogenetic,Inference, Phylogenetic,Method, Phylogenetic Comparative,Molecular Phylogenetic,Network, Phylogenetic,Phylogenetic Analyse,Phylogenetic Clusterings,Phylogenetic Comparative Analyses,Phylogenetic Comparative Method,Phylogenetic Distances,Phylogenetic Group,Phylogenetic Incongruences,Phylogenetic Inferences,Phylogenetic Network,Phylogenetic Reconstructions,Phylogenetic Relatednesses,Phylogenetic Relationship,Phylogenetic Signals,Phylogenetic Structures,Phylogenetic, Community,Phylogenetic, Molecular,Phylogenies,Phylogenomic,Reconstruction, Phylogenetic,Relatedness, Phylogenetic,Relationship, Phylogenetic,Signal, Phylogenetic,Structure, Phylogenetic,Tree, Phylogenetic
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D006913 Hydroxysteroid Dehydrogenases Enzymes of the oxidoreductase class that catalyze the dehydrogenation of hydroxysteroids. (From Enzyme Nomenclature, 1992) EC 1.1.-. Hydroxysteroid Dehydrogenase,Dehydrogenase, Hydroxysteroid,Dehydrogenases, Hydroxysteroid
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015964 Gene Expression Regulation, Bacterial Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria. Bacterial Gene Expression Regulation,Regulation of Gene Expression, Bacterial,Regulation, Gene Expression, Bacterial
D055786 Gene Knockout Techniques Techniques to alter a gene sequence that result in an inactivated gene, or one in which the expression can be inactivated at a chosen time during development to study the loss of function of a gene. Gene Knock-Out Techniques,Gene Knock Out,Gene Knock Out Techniques,Gene Knockout,Gene Knock Outs,Gene Knock-Out Technique,Gene Knockout Technique,Gene Knockouts,Knock Out, Gene,Knock Outs, Gene,Knock-Out Technique, Gene,Knock-Out Techniques, Gene,Knockout Technique, Gene,Knockout Techniques, Gene,Knockout, Gene,Knockouts, Gene,Out, Gene Knock,Outs, Gene Knock,Technique, Gene Knock-Out,Technique, Gene Knockout,Techniques, Gene Knock-Out,Techniques, Gene Knockout
D020604 Comamonas testosteroni A species of gram-negative, aerobic rods formerly called Pseudomonas testosteroni. It is differentiated from other Comamonas species by its ability to assimilate testosterone and to utilize phenylacetate or maleate as carbon sources. Pseudomonas testosteroni

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