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

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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 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 C19–27 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 3.6 kb downstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this SDRx consists of 768 bp 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.

Research highlights

► Bacteria such as Comamonas testosteroni catabolize steroids for their carbon and energy supply. ► Hydroxysteroid dehydrogenases (HSDs) are important enzymes in bacterial steroid degradation. ► A novel 7α-HSD enables Comamonas testosteroni to mineralize cholic acids. ► 7α-HSD is a novel member of the short-chain dehydrogenase/reductase (SDR) superfamily.

Introduction

Short-chain dehydrogenase/reductases (SDRs) constitute one of the largest enzyme superfamilies of NAD(P)(H)-dependent oxidoreductases which are distinct from the medium-chain dehydrogenase/reductases (MDR) and aldo-keto reductase (AKR) superfamilies [1], [2], [3]. Found in all forms of life (archaea, bacteria and eukaryotes), SDRs metabolize a range of substrates including aliphatic aldehydes and ketones, monosaccharides, steroids, prostaglandins, flavonoids, polycyclic aromatic hydrocarbons, and retinoids. Several of these SDR substrates are known to serve as important intra or intercellular signal molecules in pro and eukaryotes, which is especially true for steroids, retinoids, and flavonoids [4], [5].

Comamonas testosteroni ATCC11996 is a Gram-negative bacterium that belongs to the beta group of the Proteobacteria. These strictly aerobic, non-fermentative, chemoorgano-trophic bacteria rarely attack sugars, but grow well on organic acids and amino acids [6]. Moreover, this bacterium is able to grow on steroids or aromatic hydrocarbons as sole carbon and energy source and thus may represent an important means in the mineralization of these stable compounds. We have shown in earlier investigations that 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from C. testosteroni is a member of the SDR superfamily and catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus of a great variety of C19–27 steroids [7], [8]. In addition, 3α-HSD/CR mediates 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 [7], [9], [10]. Recently, we found that gene expression of 3α-HSD/CR in C. testosteroni ATCC11996 is controlled by two repressor proteins, RepA and RepB, at the transcriptional and translational level, respectively [11], [12].

In the present work, a novel short-chain dehydrogenase/reductase gene (SDRx) in C. testosteroni ATCC11996 was found and shown to be located 3.6 kb downstream of the hsdA gene. As a member of the SDR superfamily, this novel SDRx also contains the common sequence motifs of SDRs that define the cofactor binding site Gly-X-X-X-Gly-X-Gly in the N-terminal region and the catalytic residue motif Tyr-X-X-X-Lys. Phylogenetic analyses revealed that SDRx is a 7α-hydroxysteroid dehydrogenase (7α-HSD) that is involved in steroid metabolism. This finding was further stated by an SDRx knock-out mutant strain that showed an impaired growth on steroids.

Section snippets

Bacterial strains and plasmids

Host strains Escherichia coli HB101 (Promega) and C. testosteroni ATCC11996 (Deutsche Sammlung für Mikroorganismen) were used for cloning and gene expression. Cloning of PCR fragments was carried out in pCR2.1-TOPO (Invitrogen). For overexpression and purification of SDRx, E. coli strain BL21 (DE3) pLysS was transformed with plasmid pET-15b from Novagen.

Growth media and growth conditions

Bacterial cells were grown in a shaker (180 rpm) in Standard I Nutrient broth medium (Merck, Darmstadt) or LB medium at 37 °C (E. coli) or 27 °C (

Cloning and sequencing of the SDRx gene

In this study, a novel SDR gene, SDRx, from C. testosteroni was found to be located 3.6 kb downstream from hsdA, the latter being extensively characterized in our previous work (Fig. 1) [7], [8], [9], [10], [11], [12]. The SDRx gene contains 768 base pairs coding for a novel protein of 255 amino acids with a predicted molecular mass of 27 kDa. The nucleotide sequence and the deduced amino acids of SDRx are shown in Fig. 2. The GenBank accession number of the nucleotide sequence of SDRx gene is

Discussion

In our previous investigations, 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) has been found to be functional as oxidoreductase toward a variety of steroid substrates including the steroid antibiotic fusidic acid [22]. The enzyme also catalyzes the carbonyl reduction of non-steroidal aldehydes and ketones such as an insecticide (NKI 42255) [22]. Interestingly, 3α-HSD/CR from C. testosteroni has been found to be inducible by testosterone and progesterone. In the vicinity of the

Acknowledgements

We thank the “State-Sponsored Scholarship Program for Graduate Students” funded by the Chinese Scholarship Council for financial support. This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1704/4-1; MA 1704/4-2).

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