ORF18-disrupted mutant of Comamonas testosteroni TA441 accumulates significant amounts of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and its derivatives after incubation with steroids

In a steroid degradation gene cluster of Comamonas testosteroni TA441 consisting of ORF18, 17 and tesIHA2A1DEFG, ORF18 was implicated in encoding a CoA-transferase by database searches, but the matching substrate was not clear. In this study, ORF18 was shown to be necessary for conversion of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, a product of hydrolysis of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid in steroid degradation by TA441. The ORF18-disrupted mutant accumulates 7-hydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 7,12-dihydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid when incubated with chenodeoxycholic acid and cholic acid, respectively.     

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.     

Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30.     

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

Identification of 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid, 4-Hydroxy-2-Oxohexanoic Acid, and 2-Hydroxyhexa-2,4-Dienoic Acid and Related Enzymes Involved in Testosterone Degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

Biochemical aromatization of 2-methyleneandrostenedione: stereochemistry of hydrogen removal at the C-1 position

To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1-²H]labeled steroid 1 and its [1β-²H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography–mass spectrometry (GC–MS) in each. Parallel experiments with the natural substrates [1-²H] and [1β-²H]ADs were also carried out. The GC–MS analysis indicated that 2-methyl estrogen 2 produced from [1-²H]labeled substrate 1 retained completely the 1-deuterium (1β-H elimination), while product 2 obtained from [1β-²H]isomer 1 lost completely the 1β-deuterium. Stereospecific 1β-hydrogen elimination was also observed in the parallel experiments with the labeled ADs as established previously. The results indicate that biochemical aromatization of the 2-methylene steroid 1 proceeds through the 1β-hydrogen removal concomitant with cleavage of the C₁₀–C₁₉ bond, yielding 1(10),4-dienone 9, in a similar manner to that involved in AD aromatization. This would give additional evidence for the stereomechanisms for the last step of aromatization of AD, requiring the stereospecific 1β-hydrogen abstraction and cleavage of the C₁₀–C₁₉ bond, and for the enolization of a carbonyl group at C-3 in the A-ring aromatization.     

A New Bacterial Steroid Degradation Gene Cluster in Comamonas testosteroni TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Δ¹-dehydrogenase and 3-ketosteroid-Δ⁴(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

A new microbial degradation pathway of steroid alkaloids

In the degradation pathway of the steroid alkaloid tomatidine by Gymnoascus reesii the A-ring of tomatidine is opened with the formation of the 4-hydroxy-3,4-secotomatidine-3-oic acid, which was identified in the form of N-acetyl-3,4-tomatidine-carbolactone by mass, IR and 1H NMR spectra. Cleavage of the A-ring in the starting reaction indicates that an alternative pathway must be operating, instead of the general oxidative one.     

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.     

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.     

The genes encoding the hydroxylase of 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione in steroid degradation in Comamonas testosteroni TA441

Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB; and another consisting of ORF18, 17, tesI, H, ORF11, 12, and tesDEFG. TesH and I are, respectively, the Delta(1)- and Delta(4)(5alpha)-dehydrogenase of the 3-ketosteroid, TesD is the hydrolase for the product of meta-cleavage reaction, and TesEFG degrade one of the product of TesD. In this report, we describe the identification of the function of ORF11 (tesA2) and 12 (tesA1). The TesA1- and TesA2-disrupted mutant accumulated two characteristic intermediate compounds, which were identified as 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA) and its hydroxylated derivative, 3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione by MS and NMR analysis. A complementation experiment using a broad-host range plasmid showed that both TesA1 and A2 are necessary for hydroxylation of 3-HSA to 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA).     

Identification of genes involved in inversion of stereochemistry of a C-12 hydroxyl group in the catabolism of cholic acid by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an α-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12α-hydroxyandrosta-1,4,6-triene-3,17-dione and 12β-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441.     

FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.     

The bile acid composition of gastric contents from neonates with high intestinal obstruction.

The study was designed to identify 'atypical' bile acids in gastric contents from three neonates with high intestinal obstruction on the basis that this was likely to represent a rich source of primary bile acids. Cholic acid was the major component, and related 'atypical' bile acids included its C-3 and C-7 oxidation products, its 3 beta-epimer and 2 beta- and 6 alpha-hydroxylation products. Allocholic acid was the only 5 alpha-cholanic acid derivative identified. 7 alpha, 12 alpha-Dihydroxy-3-oxochol-4-en-24-oic acid was found in all three specimens and might be an intermediate in a biosynthetic pathway from cholesterol to cholic acid in which side-chain oxidation precedes at least some of the nuclear changes. Side-chain-hydroxylated derivatives of trihydroxycoprostanic acid were also detected and these may represent intermediates in biosynthetic pathways from cholesterol to cholic acid via 5 beta-cholestan-3 alpha, 7 alpha, 12 alpha-triol. The most abundant bile acid of this type was (25 epsilon)-3 alpha, 7 alpha, 12 alpha, 25-tetrahydroxy-5 beta-cholestan-26-oic acid, which suggested that C-25 hydroxylation may be an important step in the shortening of the C8 side chain of the cholestane triol to the C5 side chain of cholic acid in the neonatal period. Bile acids lacking a substituent at C-12 included chenodeoxycholic acid, its C-3 and C-7 oxidation products, its 3 beta-epimer and its 6 alpha-hydroxylation product (hyocholic acid).     

Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway

The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C₁₉ androgens. By using a ¹³C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C₁₇ intermediate, we propose that the A-ring cleavage may be followed by the removal of a C₂ side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (≤500 μM) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C₁₉ androgens.     

Stereospecific formation of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid from either (25R)- or (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid by rat liver homogenate

Studies of the stereochemistry of the intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, in the biosynthetic sequence between 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and cholic acid have been undertaken. (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-Trihydroxy-5 beta-cholestan-26-oic acid was incubated with rat liver homogenates. The reaction products were converted to p-bromophenacyl ester derivatives and the esters were analyzed by high-performance liquid chromatography. By comparison with authentic samples of two (24E)- and (24Z)-isomers of the alpha, beta-unsaturated acid and of four isomers at C-24 and C-25 of the beta-hydroxy acid, (24E)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid were found to be formed from either (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. No formation of the (24Z)-isomer of the trihydroxycholestenoic acid or the other three isomers of the tetrahydroxycholestanoic acid was detected. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.