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Applied and Environmental Microbiology, September 2005, p. 5275-5281, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5275-5281.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Masae Horinouchi,^1* Toshiaki Hayashi,¹ Hiroyuki Koshino,¹ Tomokazu Kurita,¹ and Toshiaki Kudo^1,2

RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,¹ Science of Biological Supermolecular Systems, Graduate School of Integrated Science, Yokohama City University, Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan²

Received 19 January 2005/ Accepted 13 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several species of bacteria, including Nocardia restrictus and Comamonas testosteroni (formerly Pseudomonas testosteroni), are known for the ability to utilize testosterone and various other steroids as sole carbon and energy sources. The mechanism by which testosterone is degraded in N. restrictus and C. testosteroni was eagerly studied, and the main intermediate compounds in the degradation pathway of these bacteria, especially N. restrictus, were determined in 1960s (2-6, 15-18). We simultaneously identified the genes and the intermediate compounds that are accumulated by gene-disrupted mutants and revealed the testosterone degradation pathway and degradation genes in Comamonas testosteroni TA441 (9-13). In TA441, the A ring of testosterone is aromatized and meta-cleaved, and the resultant 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid (4,9-DSHA) is divided into two compounds through hydrolysis by TesD. The procedure after aromatization of the A ring is similar to that of the bacterial degradation of aromatic compounds such as biphenyl, and TesD shows about 40% identity with BphD, a hydrolase in biphenyl degradation. Assuming that the reaction of TesD is similar to that of BphD, the products of TesD were predicted to be 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid, although we could not isolate and identify the products in the previous study. In this study, we isolated and identified the products of TesD in testosterone degradation with complete mass spectrometry (MS) and nuclear magnetic resonance (NMR) data. We also identified the substrate and the product of hydratase TesE, which is encoded just downstream of TesD.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of 4,9-DSHA.
The tesD-disrupted mutant was incubated in 20 ml of LB medium for about 15 h. The culture was inoculated into 500 ml of one-half LB medium plus one-half C medium with 0.1% 1,4-androstadiene-3,17-dione (ADD) and incubated at 30°C for about 15 h until the medium showed an intense yellow color. C medium is a mineral medium for TA441 to grow with steroid compounds (13). The culture was centrifuged, and the supernatant was extracted twice with the same volume of ethyl acetate. The aqueous layer was then acidified with 6 M HCl and extracted twice with the same volume of ethyl acetate. The ethyl acetate layer extracted under acidic conditions was treated with Na₂SO₄ and concentrated in vacuo. The oily residue was redissolved in a small amount of methanol and loaded onto a Waters 600 high-pressure liquid chromatography (HPLC) system (Nihon Waters, Tokyo, Japan) with an Inertsil ODS-3 column (20 by 250 mm; GL Science, Tokyo, Japan) and a mobile phase with the composition CH₃CN-CH₃OH-H₂O-trifluoroacetic acid (TFA) at a ratio of 50:10:40:0.05 and was eluted at a flow rate of 8 ml/min at 40°C. 4,9-DSHA was detected at 316 nm. About 25 ml of the fraction containing 4,9-DSHA was collected and concentrated in vacuo to about 10 ml. The concentrated fraction was used directly for the transformation by TesD.

Transformation of 4,9-DSHA by TesD.
Escherichia coli JM109 harboring pTesD, a pUC19 derivative plasmid carrying tesD, was cultured in LB medium containing 100 µg/ml ampicillin and 200 µM IPTG (isopropyl-ß-D-thiogalactopyranoside) at 30°C for about 15 h. After the incubation, 200 µM IPTG was added to the culture, and the culture was then incubated for an additional 2 hours. The cells were centrifuged, washed twice with C medium, resuspended in C medium, and disrupted by sonication. After centrifugation, the supernatant was used as a cell extract. The reaction was initiated by the addition of the cell extract prepared from about 15 ml of the E. coli culture and carried out in 20 ml phosphate buffer (final concentration, 50 mM, pH 7.5) containing the purified 4,9-DSHA at 30°C for about an hour. The resultant reaction solution was used for purifying the two products of TesD from 4,9-DSHA (X5P-1 and X5P-2).

Isolation of product X5P-1.
Twenty milliliters of the reaction solution was acidified to pH 2 with 6 M HCl and extracted twice with the same volume of ethyl acetate. The ethyl acetate layer was treated with Na₂SO₄ and concentrated in vacuo. The residue was redissolved in a small amount of methanol and loaded onto a Waters 600 HPLC with an Inertsil ODS-3 column (20 by 250 mm) and eluted at a flow rate of 8 ml/min at 40°C. The elution was carried out using a linear gradient from 30% solution A (CH₃CN-CH₃OH-TFA [95:5:0.05]) and 70% solution B (H₂O-CH₃OH-TFA [95:5:0.05]) to 65% solution A and 35% solution B over 25 min and was then maintained for 10 min and changed to 30% solution A for 5 min. X5P-1 was detected at 280 nm. About 10 ml of the fraction containing X5P-1 was collected from a single HPLC run and concentrated in vacuo to about 4 ml. The fraction was extracted twice with the same volume of ethyl acetate, and the ethyl acetate layer was treated with Na₂SO₄ for about 15 h to remove water. The ethyl acetate layer was then dried, and the purified X5P-1 (approximately 1.6 mg) was dissolved in CD₃OD immediately for further analysis.

Isolation of product X5P-2.
Twenty milliliters of the reaction solution was acidified to pH 2 with HCl and extracted twice with the same volume of chloroform, followed by extraction twice with the same volume of ethyl acetate. Both the chloroform and the ethyl acetate layers were treated with Na₂SO₄ and concentrated in vacuo, and the residues were redissolved in a small amount of methanol. The solutions were mixed together, loaded onto a Waters 600 HPLC with an Inertsil ODS-3 column (20 by 250 mm), and eluted at a flow rate of 8 ml/min at 40°C. The elution was carried out using a linear gradient from 20% solution A and 80% solution B to 65% solution A and 35% solution B over 25 min and was then maintained for 10 min and changed to 20% solution A for 5 min. X5P-2 was detected at 288 nm, and the fraction containing X5P-2 was collected from the eluent. As the fraction containing X5P-2 was not sufficiently pure for further analysis, the fraction was purified again via HPLC under the same conditions, except for the detection (at 205 nm). The fraction was extracted twice with the same volume of ethyl acetate, and the ethyl acetate layer was dried. Purified X5P-2 (approximately 9.1 mg) was dissolved in CD₃OD and analyzed by NMR and MS.

Isolation of the conversion product of X5P-1 by TesE.
X5P-1 was prepared as described above and treated with a crude extract of E. coli expressing TesE. Portions of the reaction solution were treated with 2,4-dinitrophenylhydrazine (DNPH) at appropriate intervals and analyzed by HPLC. As the products P1 and P2 were contained in all the analyzed samples, all solutions were mixed together (total of 3 ml), extracted twice with 4 ml of ethyl acetate, treated with Na₂SO₄, concentrated in vacuo, and loaded onto an HPLC. Elution was carried out using 65% solution A and 35% solution B, and compounds P1 and P2 were detected at 349 nm. Compounds P1 and P2 were collected from the eluent and purified. DNPH derivatives of the isomers of X5P-1 were isolated and characterized in a similar manner.

3D-HPLC.
All the HPLC charts presented in this paper were obtained by three-dimensional HPLC (3D-HPLC) analysis. For the 3D-HPLC analysis, a volume of methanol twice that of a sample solution (a portion of the culture or the reaction solution) was added to the solution, which was then centrifuged. The supernatant was directly injected into the HPLC system. A Waters 2690 HPLC was utilized with an Inertsil ODS-3 column (2.1 by 150 mm), and elution was carried out using a linear gradient from 20% solution A (CH₃CN-CH₃OH-TFA [95:5:0.05]) and 80% solution B (H₂O-CH₃OH-TFA [95:5:0.05]) to 65% solution A and 35% solution B over 10 min, maintained for 3 min, and then adjusted to 20% solution A for 5 min. The flow rate was 0.21 ml/min. Three-dimensional HPLC analysis of DNPH derivatives was the same except for elution (65% solution A and 35% solution B).

General experimental procedures.
For gas chromatographic MS, a GCMS-QP5050A mass spectrometer (Shimadzu, Tokyo, Japan) fitted with an HP-5MS column (0.25 mm internal diameter by 30 m, 0.25-µm film thickness; Agilent Technologies, CA) was used. Fast atom bombardment (FAB)-MS (positive-ion mode) data were recorded on a JEOL JMS-SX 102 mass spectrometer using a glycerin matrix. UV spectra were recorded with Ultrospec 3300 (Amersham Pharmacia Biotech, NJ). One-dimensional and two-dimensional NMR spectra were taken on a JNM-ECP500 or a JNM-ECA600 spectrometer (JEOL). Tetramethylsilane at 0 ppm in CDCl₃ solution and residual CD₂HOD at 3.30 ppm in CD₃OD solution were used as internal references for ¹H chemical shifts. ¹³C chemical shifts were obtained with reference to CD₃OD (49.0 ppm) or CDCl₃ (77.0 ppm) at 25°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of 4,9-DSHA and 3D-HPLC analysis of 4,9-DSHA solution treated with TesD.
Comamonas testosteroni degrades certain steroids, such as testosterone, via aromatization of the A ring followed by meta-cleavage. In previous studies, we identified two steroid degradation gene clusters in C. testosteroni TA441. One contains the meta-cleavage enzyme gene tesB, 16 open reading frames (ORFs), and the positive regulator of steroid degradation genes tesR, and the other consists of ORF18, ORF17, and tesIHA2A1DEFG (9-13). In testosterone degradation by TA441, the product of the meta-cleavage reaction, 4,9-DSHA, is degraded by the hydrolase TesD. In a previous study, we detected one of the probable products of TesD by 3D-HPLC, but the amount was too small for isolation and identification (9). The 4,9-DSHA used in the previous study was prepared from the culture of a TesD-disrupted mutant incubated with testosterone. As 4,9-DSHA is converted nonenzymatically and irreversibly to the more stable 4-aza-9,17-dioxo-9,10-secoandrosta-1,3,5(10)-triene-3-carboxylic acid in the presence of ammonium ion (9), extension of the incubation did not effectively increase the yield of 4,9-DSHA. To increase the quantity of 4,9-DSHA accumulated, ADD was used as the substrate instead of testosterone. ADD is an intermediate compound of testosterone degradation, and previous experiments had shown that gene-disrupted mutants accumulate a greater quantity of intermediate compounds when incubated with ADD than with testosterone (11). The TesD-disrupted mutant was incubated with ADD, and 4,9-DSHA was purified from 500 ml of the culture by extraction with ethyl acetate and HPLC separation. The 4,9-DSHA solution was concentrated with an evaporator, dissolved in 20 ml of phosphate buffer (final concentration, 50 mM, pH 7.5), and treated with a crude extract of TesD expressed by E. coli. The 3D-HPLC chart of the culture of the TesD-disrupted mutant incubated with ADD is shown in Fig. 1a, and the 3D-HPLC charts for purified 4,9-DSHA before and after treatment with TesD are shown in Fig. 1b and c. Figure 1a shows a greater accumulation of 4,9-DSHA than incubation with testosterone in our previous study. After treatment with TesD, two compounds, named X5P-1 and X5P-2, were detected. X5P-1 is the product detected in the previous experiment. X5P-2 was not detected in the previous experiment, probably because of its weak absorbance.

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FIG. 1. Three-dimensional HPLC analysis of the culture of TesD-disrupted mutants incubated with ADD (a), 4,9-DSHA (b), and purified 4,9-DSHA treated with TesD (c). The vertical axis indicates wavelength (nanometers), the horizontal axis indicates retention time (minutes), and the UV absorbance of each compound is represented in contour. For the analysis, a Waters 2690 HPLC was used with an Inertsil ODS-3 column (2.1 by 250 mm; GL Science), and elution was carried out using a linear gradient from 20% solution A (CH3CN-CH3OH-TFA [95:5:0.05]) and 80% solution B (H2O-CH3OH-TFA [95:5:0.05]) to 65% solution A and 35% solution B over 10 min and then maintained for 3 min and changed to 20% solution A for 5 min. Abbreviations are as follows: Ts, testosterone; 4-AD, 4-androstene-3,17-dione; 17OH-ADD, 17-hydroxy-1,4-androstadiene-3-one; 4,9-DSHA-N, 4-aza-9,17-dioxo-9,10-secoandrosta-1,3,5(10)-triene-3-carboxylic acid.

Isolation and analysis of the compound X5P-1 produced from 4,9-DSHA by TesD.
Twenty milliliters of the reaction solution containing X5P-1 was extracted with ethyl acetate and concentrated in vacuo, and the residue was redissolved in a small amount of methanol and purified using an HPLC. Purified X5P-1 (approximately 1.6 mg) was dissolved in CD₃OD immediately and analyzed by NMR and MS. The molecular formula of X5P-1 was determined to be C₆H₈O₃ from high-resolution FAB-MS data (negative) (found, m/z 127.0391 [M – H]^–; calculated, m/z 127.0395). In the UV spectrum, a maximum absorption was observed at 280 nm in methanol and acidic methanol solution (0.01 M HCl). The ¹³C NMR spectrum showed six carbon signals, including one methyl, three olefinic methine, and two quaternary carbons at 141.8 and 168.0 ppm in CD₃OD solution. The ¹H NMR spectrum indicated the presence of a sequential partial structure from a terminal methyl group at 1.78 ppm (H-6), a cis double bond, and to an adjacent olefinic methine at 6.48 ppm (H-3). The stereochemistry of the double bond was determined by the coupling constant value (J = 10.5 Hz) between H-4 and H-5. From ¹³C chemical shift values, the remaining two quaternary carbons with three oxygen atoms were assigned as C-1 at 168.0 ppm and C-2 at 141.8 ppm in the enol form of -keto carboxylic acid (7, 19). Complete NMR assignments (Table 1) were confirmed by two-dimensional pulsed-field gradient (PFG) double quantum correlation spectroscopy, PFG heteronuclear multiple quantum correlation, and PFG heteronuclear multiple-bond correlation spectral data. Based on the these spectral data, the structure of X5P-1 was determined to be (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid (Fig. 2a). NMR data indicated that X5P-1 is stable as the enol form in methanol solution. The keto form was not detected directly, although very slow exchange and partial deuteration at H-3 were observed in CD₃OD solution caused by keto-enol tautomerism. Synthetic preparation of a 4Z and 4E mixture of 2-hydroxyhexa-2,4-dienoic acid has been previously reported (14), but this is the first isolation and characterization of the pure 4Z isomer as well as the first complete identification of 2-hydroxyhexa-2,4-dienoic acid as an intermediate of testosterone degradation (2).

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TABLE 1. NMR data for X5P-1

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FIG. 2. Structure of X5P-1 [(2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid] (a) and structure of X5P-2 {9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid [IUPAC name (3aS,4S,7aS)-7a-methyl-1,5-dioxooctahydroindene-4-propanoic acid]} (b).

Isolation and analysis of the compound X5P-2 produced from 4,9-DSHA by TesD.
Twenty milliliters of the reaction solution containing X5P-2 was extracted with solvents and purified using HPLC with detection at 288 nm and then at 205 nm. Purified X5P-2 (approximately 9.1 mg) was dissolved in CD₃OD and analyzed by NMR and MS. The molecular formula of X5P-2 was determined to be C₁₃H₁₈O₄ from high-resolution FAB-MS data (positive) (found, m/z 239.1297 [M + H]⁺; calculated, m/z 239.1283). In the UV spectrum, a maximum absorption was observed at 290 nm ( = 58) in methanol. The ¹³C NMR spectrum showed one methyl carbon at 13.5 ppm, two ketone carbonyl carbons at 210.5 and 217.9 ppm, and one carboxylic carbonyl carbon at 177.4 ppm. In the ¹H NMR spectrum, a characteristic singlet methyl proton signal was observed at 1.18 ppm. These data indicated that the structure of X5P-2 is 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid [the IUPAC (International Union of Pure and Applied Chemistry) name is (3aS,4S,7aS)-7a-methyl-1,5-dioxooctahydroindene-4-propanoic acid], which is the predicted intermediate produced by cleavage of the bond between C-5 and C-10 from 4,9-DSHA (Fig. 2b). This structure, including stereochemistry, was supported by detailed NMR study, and complete NMR assignments are summarized in Table 2.

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TABLE 2. NMR data for X5P-2

Identification of the conversion product of 2-hydroxyhexa-2,4-dienoic acid (X5P-1) by TesE.
In the previous study of TesD, we showed that one of the products of TesD was converted by TesE. This product of TesD has been identified as 2-hydroxyhexa-2,4-dienoic acid (X5P-1) in this study. As the product of TesE was not detected by HPLC in the previous study, we prepared a larger quantity of 2-hydroxyhexa-2,4-dienoic acid for conversion by TesE. 2-Hydroxyhexa-2,4-dienoic acid was purified as described above (the 3D-HPLC chart is shown in Fig. 3a to c). The purified 2-hydroxyhexa-2,4-dienoic acid was treated with TesE prepared in the same manner as TesD, and the reaction solution was analyzed by 3D-HPLC at appropriate intervals (Fig. 3d to f). 2-Hydroxyhexa-2,4-dienoic acid was converted quickly, but no product was detected. The hydrophilic character and low stability, together with the weak UV absorption of 4-hydroxy-2-oxohexanoic acid, the conjectured product of TesE, predicted its difficulty of the isolation in intact form, so the classical DNPH derivatization method was applied. Figure 3g shows the 3D-HPLC chart of 2,4-dinitrophenylhydrazine-treated 2-hydroxyhexa-2,4-dienoic acid solution right after the addition of TesE. 2-Hydroxyhexa-2,4-dienoic acid was detected at a retention time of 2.7 min together with three DNPH derivatives (A, B, and C [C is not visible in Fig. 3g]). These compounds were isolated and characterized as the E isomer (A) and Z isomer (B) of (4Z)-2-(2,4-dinitrophenylhydrazono)hex-4-enoic acid and a nonenzymatic product, 2-oxohex-3-enoic acid, obtained as a single isomer (C) (Fig. 4). NMR data of these compounds are shown in Table 3. These compounds were DNPH derivatives of predicted isomers of 2-hydroxyhexa-2,4-dienoic acid (similar spontaneous transformations are reported with other compounds [cf. reference 15]). Treatment of X5P-1 by TesE and following derivatization of DNPH led to the detection and isolation of two products, P1 and P2 (3D-HPLC charts in Fig. 3h to j). The chemical structures of P1 and P2 were determined to be DNPH derivatives of 2-oxo-4-hexanolactone by NMR analyses (Table 3) together with high-resolution FAB-MS and UV spectral data. Low field chemical shift values at 4.80 ppm of H-4 suggested the lactone structure. The geometric stereochemistry of the DNPH portions of P1 and P2 were determined to be E and Z by ¹³C chemical shift values of C-3 at 30.9 and 34.2 ppm, respectively (Fig. 4).

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FIG. 3. Three-dimensional HPLC analysis of purified 4,9-DSHA (a); purified 4, 9-DSHA treated with TesD (b); purified 2-hydroxyhexa-2,4-dienoic acid (c); purified 2-hydroxyhexa-2,4-dienoic acid right after addition of TesE (d), 30 min after addition of TesE (e), and 60 min after addition of TesE (f); and purified 2-hydroxyhexa-2,4-dienoic acid solution right after addition of TesE followed by treatment with DNPH (g), 30 min after addition of TesE followed by treatment with DNPH (h), 60 min after addition of TesE followed by treatment with DNPH (i), and 120 min after addition of TesE followed by treatment with DNPH (j). HPLC conditions and abbreviations are the same as those described in the legend of Fig. 1 for a to f and the same as those described in the legend of Fig. 1 except for elution (65% solution A and 35% solution B, constant) for g to j.

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FIG. 4. Degradation pathway of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid in steroid degradation by Comamonas testosteroni TA441 with structures of DNPH derivatives identified in this study (A, B, C, P1, and P2). Using P1 and P2, presented as authentic, 2-hydroxy-4-hex-2-enolactone was identified as the product of TesE from the reaction solution without treatment with DNPH. Isolation of 2-hydroxy-4-hex-2-enolactone indicates that the direct product of the TesE reaction is 4-hydroxy-2-oxohexanoic acid, which was considered to be converted to 2-hydroxy-4-hex-2-enolactone under the acidic conditions of the isolation procedure. Reactions of TesG and TesF and compounds in brackets are speculation. CoA, coenzyme A.

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TABLE 3. NMR data for P1, P2, A, B, and Ca

Using DNPH derivatives P1 and P2, presented as authentic, practically, HPLC purification led to the isolation of 2-hydroxy-4-hex-2-enolactone as an intact product of TesE, which is the enol form of 2-oxo-4-hexanolactone (Fig. 4). The structure of 2-hydroxy-4-hex-2-enolactone was determined from the following spectroscopic data. The molecular formula was determined to be C₆H₈O₃ from high-resolution FAB-MS data (negative) (found, m/z 127.0386 [M – H]^–; calculated, m/z 127.0396). In the UV spectrum, a maximum absorption was observed at 230 nm ( = 1,623) in methanol (0.01 M HCl). The ¹H NMR data are shown in Table 4. Isolation of 4-hydroxy-2-oxohexanoic acid did not succeed because of the use of acidic HPLC conditions for separation under which the five-member lactone ring is more stable.

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TABLE 4. 1H NMR data for TesE-P

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gram-negative bacterium Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring. The ring cleavage procedure and the enzymes involved in it are similar to those of a common bacterial aromatic compound degradation pathway. In our previous paper concerning TesD (9), a hydrolase of the product of the meta-cleavage reaction in steroid degradation, we speculated that the function of TesD would be similar to that of BphD, the corresponding enzyme in biphenyl degradation, based on the approximately 40% identity of TesD to some BphDs and the accumulation of 4,9-DSHA by a TesD-disrupted mutant. In this study, 4,9-DSHA was purified from the culture of a TesD-disrupted mutant incubated with ADD and treated with E. coli-expressed TesD, and two products (X5P-1 and X5P-2) were directly purified and identified from the reaction solution. Compound X5P-1 had six carbons and stronger UV absorption and was identified as 2-hydroxyhexa-2,4-dienoic acid by NMR and MS analysis. 2-Hydroxyhexa-2,4-dienoic acid was proposed as an intermediate compound in steroid degradation by the gram-positive bacterium Nocardia restrictus in 1966 based on the isolation of the 2,4-dinitrophenylhydrazone derivative of 2-oxo-4-hexanolactone as a conversion product of 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione treated with a cell extract of N. restrictus (6). In this study, intact 2-hydroxyhexa-2,4-dienoic acid was successfully isolated and identified from complete NMR and MS data. This is the first report of the identification of 2-hydroxyhexa-2,4-dienoic acid as an intermediate compound in testosterone degradation by C. testosteroni and the first direct identification of intact 2-hydroxyhexa-2,4-dienoic acid as a product of a steroid degradation enzyme. The isolated 2-hydroxyhexa-2,4-dienoic acid was the pure 4Z isomer, which had not been reported before. The purified 2-hydroxyhexa-2,4-dienoic acid was then treated with TesE, which is encoded downstream of TesD. The product was not detected by direct analysis of the reaction solution by 3D-HPLC, but 2-hydroxy-4-hex-2-enolactone was successfully identified. The direct product of TesE should be 4-hydroxy-2-oxohexanoic acid, which should have been lactonized to 2-hydroxy-4-hex-2-enolactone during purification under acidic conditions. The result showed TesE to be a hydratase of 2-hydroxyhexa-2,4-dienoic acid to 4-hydroxy-2-oxohexanoic acid. This reaction is the same as a step in the bacterial degradation of an aromatic compound, 4-methylcatechol (1). TesE shows approximately 60% identity to some hydratases which convert 2-hydroxypent-2,4-dienoate to 4-hydroxy-2-oxopentanoic acid in biphenyl degradation, and the following TesF and TesG show higher identity, approximately 80%, with BphI and BphJ, respectively, the corresponding enzymes in biphenyl degradation. BphJ converts 4-hydroxy-2-oxopentanoic acid into pyruvate and acetaldehyde, and the latter is converted to acetyl coenzyme A by BphI (8). Based on the high homology of TesG and TesF to BphJ and BphI, TesG is thought to degrade 4-hydroxy-2-oxohexanoic acid into pyruvate and propionaldehyde, and the latter is thought to be converted to propionyl coenzyme A by TesF. Pyruvate and propionaldehyde were found as intermediate compounds in steroid degradation by N. restrictus (6).

X5P-2, another product of TesD, was identified as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid [IUPAC name (3aS,4S,7aS)-7a-methyl-1,5-dioxooctahydroindene-4-propanoic acid]. This compound was isolated as an intermediate compound of ADD degradation by N. restrictus in 1963 with partial NMR data (17). Nocardia restrictus is a gram-positive bacterium and C. testosteroni is gram negative, and they do not show any particular genetic relation, but our results indicated that they degrade steroids by a similar pathway. Comparison of steroid degradation genes of both bacteria would be quite interesting, but at present, the genetic information for N. restrictus is not available.

In this study, we identified the function of TesD and TesE experimentally, which had been predicted only by the similarity to corresponding enzymes in biphenyl degradation and by intermediate compounds found in steroid degradation by N. restrictus. These results lend strong support to the function of TesF and TesG being as predicted by the homology search. The result of this study is summarized in Fig. 4. To clarify the whole steroid degradation in C. testosteroni, characterization of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation genes will be most important.

 
This work was partly supported by a grant from the Eco Molecular Sciences Research Program of RIKEN. M.H. was supported by a grant from the Special Postdoctoral Researchers Program of RIKEN.

 
* Corresponding author. Mailing address: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Phone: 81 48 467 9545. Fax: 81 48 462 4672. E-mail: masae{at}postman.riken.go.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
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Applied and Environmental Microbiology, September 2005, p. 5275-5281, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5275-5281.2005
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