Regulation of testosterone degradation in Comamonas testosteroni

Recently, we have identified a gene encoding a LuxR-type factor, TeiR (Testosterone-inducible Regulator), which positively regulates steroid degradation in Comamonas testosteroni. Herein, we demonstrate that TeiR interacts in vivo with steroid catabolic gene promoters. The presence of testosterone induces a significant TeiR protein increase at the early logarithmic phase of growth. Interestingly, it is not until the early stationary phase where the activation of a steroid-inducible gene promoter is observed, indicating that testosterone might not be the true inductor of the steroid degradation pathway. In addition, beta-galactosidase expression driven by a testosterone-inducible promoter is prematurely activated in cells cultured in medium supplemented with ethyl acetate extracts obtained from the early stationary phase cell-free supernatants of C. testosteroni grown in presence of testosterone. Complementation experiments of C. testosteroni wild type performed with teiR deletion constructs indicate that extra-copies of deleted-TeiR exert a dominant negative effect on the wild-type TeiR protein. While, when C. testosteroni teiR mutants were used to carry out complementation assays only the full length gene can overcome the teiR mutant phenotype. Altogether these findings indicate that TeiR regulates steroid catabolic genes interacting with their promoters and suggest that this interaction requires the presence of a testosterone-derived metabolite to induce the system.     

A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.     

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.     

A model on the regulation of 3α-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonastestosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3α-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichiacoli. Rep1 does not bind to DNA but may bind to 3α-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3α-HSD/CR in C.testosteroni by steroids in fact appears to be a de-repression, where the steroidal ‘inducer’ prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.     

Understanding oligomerization in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: an in silico approach and evidence for an active protein

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni belongs to the short chain dehydrogenase/reductase (SDR) protein superfamily and catalyzes the oxidoreduction of a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also mediates the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to the bioremediation of natural and synthetic toxicants by C. testosteroni. Crystallization and structure analysis showed that 3alpha-HSD/CR is active as a dimer. Dimerization takes place via an interface axis which has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure. For example, 3alpha/20beta-HSD from Streptomyces hydrogenans exhibits two main subunit interfaces arranged about two non-crystallographic two-fold axes which are perpendicular to each other and referred to as P and Q. This mode of dimerization is, however, sterically impossible in 3alpha-HSD/CR because of a 28 amino acids insertion into the classical Rossmann-fold motif between strand betaE and helix alphaF. This insertion is masking helices alphaE and alphaF, thus preventing the formation of a four helix bundle and enables the dimerization via a P-axis interface. This type of dimerization in SDRs has never been observed in a crystal structure so far. The aim of this study was to investigate whether the lack of this predominantly alpha-helical subdomain keeps 3alpha-HSD/CR to be an active enzyme and whether, by an in silico approach, the formation of a homotetramer or even a novel oligomerization mode can be expected. Redesign of this interface was performed on the basis of site directed mutagenesis and according to other SDR structures by an approach combining "in silico" and "wet chemistry". Simulations of sterical and structural effects after different mutations, by applying a combination of homology modelling and molecular dynamic simulations, provided an effective tool for extensive mutagenesis studies and indicated the possibility of tetramer formation of truncated 3alpha-HSD/CR. In addition, despite lacking the extra loop domain, mutant 3alpha-HSD/CR was shown to be active towards a variety of standard substrates.     

Steroid degradation gene cluster of Comamonas testosteroni consisting of 18 putative genes from meta-cleavage enzyme gene tesB to regulator gene tesR

Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB and ORF1, 2, 3; and another consisting of ORF18, 17, tesI, H, A2, and tesA1, D, E, F, G (tesA2 to ORF18 and tesA1 to tesG are encoded in opposite directions). Analysis of transposon mutants with low steroid degradation revealed 13 ORFs and a gene (ORF4, 5, 21, 22, 23, 25, 26, 27, 28, 30, 31, 32, 33, and tesR) involved in steroid degradation in the downstream region of ORF3. TesR, which is almost identical to that of TeiR, a positive regulator of Delta1-dehydrogenase (corresponds to TesH in TA441) and 3alpha-dehydrogenase (currently not identified in TA441), in C. testosteroni ATCC11996 (Pruneda-Paz, 2004), was shown to be necessary for induction of the steroid degradation gene clusters identified in TA441, tesB to tesR, tesA1 to tesG, and tesA2 to ORF18. At least some of the ORFs from ORF3 to ORF33 were suggested to be involved in 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation.     

Extraction of a steroid transport system from Pseudomonas testosteroni membranes and incorporation into synthetic liposomes

A steroid binding protein has been extracted from Pseudomonas testosteroni membranes with an organic solvent system. This protection binds some C19 and C21 steroids but not C18 steroids. When this protein is incorporated into synthetic lipid vesicles constructed from P. testosteroni phospholipids, the vesicles perform concentrative uptake of testosterone in the presence of the ionophore valinomycin. This steroid binding protein is thus believed to be the steroid permease of this organism.     

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5

Natural and synthetic steroid hormones excreted into the environment are potentially threatening the population dynamics of all kinds of animals and public health. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. 16S-rRNA analysis showed that bacterial strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the (3)H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5 h and 20 h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in adaptive steroid degradation in Comamonas testosteroni (C. testosteroni), in previous investigations, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established. By this meta-genomic system, two estradiol inducible genes coding 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively, which are involved in steroid degradation, were found in marine strain H5. In the present work, the 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli (E. coli) strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with isopropyl-β-D-thiogalactoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one step using nickel-chelate chromatography. After protein determination, 3-ketosteroid-delta-1-dehydrogenase (0.48 mg/ml) and carboxylesterase (1.28 mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In summary, we have shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA, and purified the corresponding proteins for further research. The exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. The strain might be used for the bioremediation of steroid contaminations in seawater.     

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.     

Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.     

Characterization of the steroid degrading bacterium S19-1 from the Baltic Sea at Kiel, Germany

Steroid contamination of sea water is an ever growing problem and impacts population dynamics of all kinds of sea animals. We have long experience with the soil bacterium Comamonas testosteroni ATCC11996 which is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons, and which might be used in the bioremediation of contaminated places. In C. testosteroni about 20 enzymes which are involved in steroid degradation could be induced by 0.5 mM testosterone. In this work, we isolated a marine bacterial strain, S19-1, from the Baltic Sea at Kiel (Germany) which degrades steroids and is able to use steroids as carbon source. S19-1 was characterized as being Gram negative, and 16S rRNA analysis showed that it belongs to the genus Buttiauxella of the Enterobacteriaceae family. Strain S19-1 could be best grown in SIN medium supplemented with 0.6-4.1% NaCl at 20°C. As a carbon source, testosterone, estradiol or cholesterol in minimal medium could be used by S19-1. Moreover, S19-1 could grow up in medium with 50 μg/ml erythromycin. Plasmids pK18 and other plasmids were successfully transformed into S19-1 by chemical transformation (CaCl(2)) and shown to replicate in the cells. Currently, work is ongoing to find the steroid inducible genes.