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Applied and Environmental Microbiology, August 2007, p. 4776-4784, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00329-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Degradation Pathway of Bisphenol A: Does ipso Substitution Apply to Phenols Containing a Quaternary {alpha}-Carbon Structure in the para Position?{triangledown} ,{dagger}

B. Kolvenbach,1 N. Schlaich,2 Z. Raoui,1,2 J. Prell,3 S. Zühlke,4 A. Schäffer,1 F. P. Guengerich,5 and P. F. X. Corvini6*

Department of Environmental Research, Rheinisch-Westfälische Technische Hochschule (RWTH), Aachen University, D-52074 Aachen, Germany,1 Department of Plant Physiology, RWTH Aachen University, D-52074 Aachen, Germany,2 School of Biological Sciences, University of Reading, Reading, Berkshire RG6 6AJ, United Kingdom,3 Institute of Environmental Research (INFU), University of Dortmund, D-44221 Dortmund, Germany,4 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146,5 Institute for Ecopreneurship, University of Applied Sciences Northwestern Switzerland, CH-4132 Muttenz, Switzerland6

Received 9 February 2007/ Accepted 1 June 2007


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ABSTRACT
 
The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary {alpha}-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary {alpha}-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an 18O2 atmosphere were performed. One atom of 18O2 was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary {alpha}-carbon.


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INTRODUCTION
 
With a worldwide production amounting to more than two million tons in 2003, 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, commonly named bisphenol A (BPA), is one of the highest-volume chemicals produced (1). Due to its toxicity at low doses and its effects on the endocrine system, BPA is a public health concern (4, 19, 40). Microbial degradation of BPA leads to metabolites such as 4,4'-dihydroxy-{alpha}-methylstilbene, 2,2-bis(4-hydroxyphenyl)-1-propanol, 2,2-bis(4-hydroxyphenyl)propanoic acid, and 2-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)propane (32, 37). In fungi, lignin-degrading enzymes such as manganese peroxidase and laccase are mainly involved in the biodegradation of BPA to polymerization products 4-isopropylphenol, 4-isopropenylphenol, and hexestrol (17, 24).

To date, only catabolic pathways starting with the oxidation of BPA to a phenonium ion intermediate have been reported (Fig. 1A). These pathways were originally described for the unidentified strain MV1 (21) and have been reported for other bacteria, all belonging to the sphingomonads, e.g., Sphingomonas sp. strain WH1 (31), Sphingomonas sp. strain AO1 (33), recently designated as S. bisphenolicum (28), and S. paucimobilis strain FJ4 (18). Degradation of BPA by strain MV1 occurs via several rearrangements involving phenonium ion intermediates (21, 35). Solvolysis of the first phenonium ion intermediate (Fig. 1A, compound 1) leads to the formation of 2,2-bis(4-hydroxyphenyl)-1-propanol (Fig. 1A, compound 2) and mainly to the production of the rearranged 1,2-bis(4-hydroxyphenyl)-2-propanol (Fig. 1A, compound 3). The mechanism of oxidation of BPA with a simultaneous rearrangement of the phenonium ion has not yet been elucidated. Nonetheless, the presence of a quaternary {alpha}-carbon in the molecule probably plays an important role in the C-C bond breakage during the rearrangement process. The 1,2-bis(4-hydroxyphenyl)-2-propanol is further dehydrated into 4,4'-dihydroxy-{alpha}-methylstilbene. The stilbene intermediate is degraded into hydroxybenzaldehyde and 4-hydroxyacetophenone, which are able to support bacterial growth. 2,2-bis(4-Hydroxyphenyl)-1-propanol is further oxidized into 2,2-bis(4-hydroxyphenyl)propanoic acid and the rearranged product 2,3-bis(4-hydroxyphenyl)-1,2-propanediol (Fig. 1A, compound 6) via the production of a second phenonium intermediate (Fig. 1A, compound 4). The 2,3-bis(4-hydroxyphenyl)-1,2-propanediol is degraded very slowly into 4-hydroxybenzoic acid and 4-hydroxyphenacyl alcohol. Both reactions that lead to rearrangements are catalyzed by the same enzymatic reaction. Recently, evidence for similar rearrangements was reported for Sphingomonas sp. strain AO1, where 4,4'-dihydroxy-{alpha}-methylstilbene, 4-hydroxyacetophenone, 1,2-bis(4-hydroxyphenyl)-2-propanol, and 2,2-bis(4-hydroxyphenyl)-1-propanol were also detected as metabolites of BPA (33). In this strain, a P450 cytochrome is involved in the first degradation step, and both NADH and NADPH are used as cofactors, while flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) have no effect on the reaction (32, 33). 4-Hydroxyacetophenone and 4-hydroxybenzoic acid were also detected as metabolites of BPA in cultures of Sphingomonas sp. strain WH1 (31).


Figure 1
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  		FIG. 1. Molecular rearrangements during the degradation of BPA and NP. Dashed arrows indicate further degradation of the compounds as the source of carbon. (A) Rearrangement reactions in the catabolic pathways of BPA in strain MV1 (35). 1, phenonium ion intermediate; 2, 2,2-bis(4-hydroxyphenyl)-1-propanol; 3, rearranged 1,2-bis(4-hydroxyphenyl)-2-propanol; 4 and 5 are postulated intermediates; 6, rearranged 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. (B) ipso substitution, NIH shift, and Baeyer-Villiger molecular rearrangements during the metabolism of NP in Sphingomonas sp. strain TTNP3 (6). 1, quinol intermediate; 2, HQ; 3, postulated carbocationic intermediate; 4, alkyloxyphenol; 5, alkylbenzenediol; 6, nonanol. Central pathway, HQ is formed as the result of ipso-hydroxylation and detachment of the carbocationic intermediate; the nonanol is the result of solvolysis of the carbocationic intermediate by a molecule of water. Side-reactions, the alkylbenzenediol results from an intramolecular NIH shift or from an electrophilic substitution of one atom of hydrogen by the carbocationic intermediate in the de novo-produced HQ; the alkoxyphenol is formed via a 1,2-C,O shift (14) or via the electrophilic addition of the carbocationic intermediate to the de novo-produced HQ (6).

Recently, the metabolism of nonylphenol (NP) has been reviewed (5). Investigations were carried out mainly with Sphingomonas sp. strain TTNP3 (6-11) and S. xenophaga strain Bayram (13, 14). In both strains, degradation pathways involve ipso-hydroxylation of NP as the first step (Fig. 1B) and proceed via an ipso substitution (type II) in Sphingomonas sp. strain TTNP3 (5, 6). ipso substitutions are grouped into two types according to the nature of the substituent eliminated from the quinol (27). In type I, the substituent is eliminated as an anion, and p-benzoquinone is formed. In type II, the substituent leaves the molecule as a carbocation, and hydroquinone (HQ) is the degradation intermediate. In this unusual C-C bond-breakage reaction, which is unique in microorganisms, the presence of a quaternary {alpha}-carbon was assumed to be a prerequisite for the release of the alkyl chain as a tertiary carbocation intermediate (Fig. 1B, compound 3). It was assumed that the latter reacts further with surrounding water to form nonanol (Fig. 1B, compound 6). The central metabolite of the reaction is in fact HQ (Fig. 1B, compound 2), and previously reported side reactions such as Baeyer-Villiger rearrangement and an NIH shift involving the carbocationic intermediate lead to the production of alkyloxyphenol (Fig. 1B, compound 4) and alkyl benzenediol (Fig. 1B, compound 5) as terminal metabolites (7, 8). Both NP and BPA belong to the alkylphenol family and share a common structural feature, i.e., a quaternary {alpha}-carbon.

The present study provides evidence for a novel degradation pathway of BPA, including new metabolites. The transformation of [U-ring-14C]-labeled BPA and [U-ring-14C]-labeled NP isomer in resting cell suspensions and cell extracts of Sphingomonas sp. strain TTNP3 is reported here.


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MATERIALS AND METHODS
 
Radiochemicals.
[U-ring-14C]-labeled BPA (Hartmann Analytic, Germany) with a specific radioactivity of 1.08 GBq/mmol was diluted with nonradioactive BPA to a radioactive specificity of 96 MBq/mmol.

The 14C-labeled isomer of NP, i.e., 4-[1-ethyl-1,3-dimethylpentyl]phenol (p353NP) with a specific radioactivity of 110 MBq/mmol was prepared by Friedel-Crafts alkylation from [U-ring-14C]phenol and nonlabeled phenol with 3,5-dimethyl-3-heptanol and with BF3 as the catalyst and purified on thin-layer chromatography plates as described elsewhere (39). 14C-labeled NP was not diluted with nonradioactive NP. BPA and NP were dissolved in methanol before use.

Resting cell suspensions.
Cultures of Sphingomonas sp. strain TTNP3 were prepared on standard I medium as reported previously (10). At the late growth phase, cells were harvested for the preparation of the resting cell suspension as previously described (6). Briefly, cultures were centrifuged and pellets were resuspended in 50 mM phosphate buffer (pH 7.0). This washing procedure was carried out three times. After the last centrifugation step, cells were resuspended in the same phosphate buffer to a defined biomass (dry weight) per ml of buffer.

Cell extracts.
Cell extracts were prepared by concentrating cells in late exponential growth phase at a biomass dry weight concentration of 13 mg/ml in 5 ml of phosphate buffer. Lysozyme was added to the cell suspension at a final concentration of 1 mg/ml. After the digested cells were kept on ice for 20 min, 4-(2-aminoethyl)-benzenesulfonyl fluoride was added at a final concentration of 2 mM as the protease inhibitor, and the suspension was loaded into a French press (SLM Aminco, Rochester, NY) fitted with a 3.7-ml cell. Unless stated otherwise, dithiothreitol and FAD were added to the cell extract at a concentration of 1 mM and 1.23 mM, respectively, after five lysis cycles at 138 MPa. The cell extract was centrifuged at 10,000 x g for 10 min before being filtered through 0.2-µm pore filters. For assays carried out with various combinations of cofactors, cell lysis was achieved by ultrasonicating the cell suspension (70 W, 0.2-s pulse) for 40 min at 4°C by means of a Labsonic U device (Braun Diessel Biotech, Melsungen, Germany). Protein concentration was determined according to the method of Lowry (22).

Degradation activity of resting cells.
For degradation experiments with resting cells of Sphingomonas sp. strain TTNP3, BPA and NP were added to glass vessels at a concentration of 100 µM each; the final radioactivity was 3.8 kBq and 4.4 kBq, respectively. The solvent was removed under a gentle stream of nitrogen before starting the reaction by the addition of 400 µl of the resting cell suspension (5 mg of cells [dry weight]/ml). The mixture was incubated for 30 min under continuous magnetic stirring at 37°C. Reactions were stopped by acidifying the reaction mixture with HCl (to pH 2 to 3) and adding an equal volume of methanol to the sample. In order to obtain a maximal dissolution of the parent compounds, 20 µl of an aqueous Brij 58 (Applichem, Darmstadt, Germany) solution (13% wt/vol) was added. Before high-performance chromatography (HPLC) analysis, cells were centrifuged out, and remaining cells were removed by means of 0.2-µm-pore-sized filtration.

Enzymatic assays.
Cell extracts were assayed for monooxygenase activity using a method described elsewhere (23). The substrate BPA or NP was applied as described above for resting cell assays. Various cofactors were added separately and in combination at a concentration of 1 or 20 mM for NADPH and NADH and 0.1 or 1 mM for FAD and FMN. The final reaction mixture volume was 400 µl. The degradation assays were started by the addition of 313 µl of the cell extract. The assays were incubated for 1 h with stirring at 37°C. Sample preparation was carried out as described above for resting cells.

For inhibition tests, metyrapone [2-methyl-1,2-di-(3-pyridyl)-1-propanone], proadifene (2-DEAE-2,2-diphenylvalerate, also termed SKF-525A), and octylamine were tested at a final concentration of 3 mM or 5 mM. Remaining concentrations of BPA during inhibition assays were determined by means of gas chromatography-mass spectrometry (GC-MS).

Transposon mutagenesis.
Transposon (Tn) mutagenesis in Sphingomonas sp. strain TTNP3 was carried out by conjugational transfer of vector pSUP1021 bearing transposon Tn5 into this strain (34). The Escherichia coli S17-1 donor strain containing pSUP1021 and the recipient Sphingomonas strain were grown to exponential phase, pelleted, and resuspended in Luria-Bertani (LB) medium. A 1:2 mixture of donor and recipient was incubated on a nitrocellulose membrane (0.2-µm-pore size) on an LB agar plate overnight at 30°C. The cells were resuspended in LB medium and plated on selective LB medium containing 300 µg/ml streptomycin (selecting Sphingomonas) and 25 µg/ml kanamycin. Plates were incubated for 2 to 4 days until kanamycin-resistant colonies appeared. Growing colonies were individually picked from both LB medium containing 25 µg/ml kanamycin and agar mineral medium (36) containing 25 µg/ml kanamycin and 1 mg/ml NP. Due to the formation of an emulsion of NP with the solid medium, the latter was opaque, and as a consequence of the NP degradation, the agar matrix became clear around the colonies. The three clones that failed to produce such a halo in the NP plates were isolated correspondingly from the LB plates. After cultivation on HQ containing agar mineral medium, one NP mutant colony remained and was further used for the preparation of resting cell suspension and cell extracts.

Purification of metabolites.
In order to produce a sufficient amount of BPA metabolites, 12 ml of a resting cell suspension of Sphingomonas sp. strain TTNP3 (15 mg of cells [dry weight]/ml) was incubated with 100 µM 14C-labeled BPA for 45 min at 37°C. The assay was acidified to pH 2 to 3 by adding few drops of H2SO4. The mixture was extracted three times with 10 ml of ethyl acetate, with vigorous shaking. The combined organic phase was filtered over Na2SO4 on filter paper and dried under a gentle stream of nitrogen gas. The residue was completely resuspended in 1 ml of HPLC-grade acetonitrile. Aliquots (50 µl) of the acetonitrile extract were injected repeatedly (16 injections) into the HPLC system as described in the next section. Radioactive compounds eluted from HPLC were collected individually in flasks containing acetonitrile, with an excess of Na2SO4 to dry the samples rapidly. The samples were concentrated to dryness under a stream of nitrogen and resuspended in 100 µl of acetonitrile. The isolated compounds were further analyzed using GC-MS and a liquid chromatography-mass spectrometry (LC-MS) radiodetector. Solutions of the reference compounds HQ and isopropylphenol were prepared in acetonitrile at a concentration of 0.95 mg/liter.

Degradation assays with 18O2.
Degradation assays of BPA and NP in the presence of 18O2 were carried out with resting cells (NP) or cell extracts (BPA and NP). Nonradiolabeled BPA and NP were applied in glass vessels. The recipients were hermetically closed with rubber septa and aluminum seals. The oxygen and the solvent of the substrate solution were removed using a continuous stream of a mixture of argon and nitrogen for 5 min. For assays containing cell extracts, 250 µl of 120 mM NADPH and 75 µl of 20 mM MgSO4 (both dissolved in phosphate buffer) were added. The flasks were charged with an 18O2 atmosphere (Sigma-Aldrich, Taufkirchen, Germany). Resting cells (800 µl) and 1,200 µl of phosphate buffer or 1,200 µl of cell extract containing 1.3 mM FAD were added, and the glass vials were tightly closed. The vessels were incubated at 37°C for 30 min for the assays containing resting cells or for 1 h for assays containing cell extract. During the first 3 min of incubation, 18O2 was supplied. The reaction was stopped by acidifying the solution to pH 2 to 3 with H2SO4 and extracting three times with 2 ml of ethyl acetate with vigorous shaking. Ethyl acetate extract was dried over Na2SO4 concentrated to approximately 200 µl under a stream of nitrogen. Controls were carried out under natural 16O2 atmosphere in parallel.

HPLC analysis of reaction mixtures.
Samples were analyzed using an HPLC series 1100 (Agilent Technologies, Germany) equipped with an autoinjector, a degasser, a diode array detector, and an online liquid scintillation radioflow detector (Ramona Star; Raytest, Straubenhardt, Germany) with a cell volume of 1,300 µl. The flow rate of the scintillation cocktail (Quicksafe Flow 2; Zinsser Analytic GmbH, Frankfurt, Germany) was 2.0 ml min–1. Prior to analysis, samples were centrifuged at 15,000 x g for 20 min, and 50 µl of supernatant was injected onto a Nucleosil octadecylsilane column (4.6 mm by 150 mm, 5-µm particle size; Agilent Technologies). The compounds were eluted with a gradient of water (A) and acetonitrile (B) (both HPLC-grade; Roth, Germany) as follows: 25% B in A with a linear gradient to 90% B during 22 min, after 90% B isocratic for 5 min. Finally, the system returned to its initial conditions (25% B in A) within 10 min and was maintained for 3 min before the next run started. The UV signal was measured at 270 nm.

LC-MS radiodetector analysis.
The HPLC analyses of radioactive samples were performed as reported previously (3) with a ThermoFinnigan surveyor LC pump. Compounds were separated on a Luna C18 100-Å column (150 mm by 4 mm, 3-µm particle size) from Phenomenex (Torrance, CA). The mobile phase consisted of water (A) and acetonitrile (B). Samples were separated using a gradient program as follows: 20% B in A with a linear gradient to 30% B during 3 min, and to 100% B during another 18 min. After 100% B isocratic for 6 min, the system returned to its initial conditions (20% B in A) within 1 min and was kept in this composition for 8 min before the next run was started. After splitting (1:1) of the solvent flow (flow rate of 800 µl/min), compounds were detected simultaneously via a radioisotope detector (Raytest; Germany) with a 3139 quartz cell and a TSQ Quantum Ultra AM tandem mass spectrometer (Thermo Finnigan). Full-scan mass spectra were obtained using TSQ Quantum equipped with an atmospheric pressure chemical ionization ion source (Ion Max) operating in the negative mode over the m/z range of 92 to 500. Nitrogen was employed as both the drying and the nebulizer gas. Product ion spectra were obtained after fragmentation at collision energies of 10, 20, 30, 44, and 55 V.

GC-MS analyses.
Unless stated otherwise, 50-µl samples were thoroughly mixed with 50 µl of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA; Merck, Germany) and afterwards derivatized at 70°C for 15 min prior to GC-MS analysis. The GC-MS studies were carried out with an HP 5890 series II gas chromatograph (Agilent Technologies) equipped with an FS-SE-54-NB-0.5 column (25 m by 0.25 mm, 0.46-µm film thickness; CS Chromatographie Service, Germany) coupled to an HP 5971A mass selective detector (Agilent Technologies) as described previously (39).


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RESULTS
 
Transformation of BPA by resting cell suspensions of Sphingomonas sp. strain TTNP3.
Whether BPA is degraded by Sphingomonas sp. strain TTNP3 was investigated by incubating resting cells in the presence of [U-ring-14C]-labeled BPA (100 µM). The analysis of the supernatants by HPLC/radiodetection showed a marked decrease of the BPA peak within 30 min of incubation, and many new compounds were formed (Fig. 2). In order to identify the other compounds, various fractions were collected for further analysis by means of GC-MS and/or LC-MS/radiodetection.


Figure 2
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  		FIG. 2. Evidence from HPLC/radiodetection for the degradation of BPA and the formation of metabolites in a resting cell assay. Resting cells of Sphingomonas sp. strain TTNP3 were incubated with 100 µM [U-ring-14C]-labeled BPA for 30 min at 37°C. Arrow indicates the peak corresponding to BPA. Peak numbering is used for describing the identification of the metabolites in the text.

Identification of HQ.
On the basis of previous experiments carried out with NP as substrate, in which HQ was formed as a metabolite, one compound contained in the first collected fraction (Fig. 2, peak 2) was proposed to be HQ (Fig. 3, compound 3). The GC-MS analysis of the derivatized fraction (trimethylsilylation) showed a molecular ion at m/z 254 (Table 1). The metabolite could be definitely identified as HQ by comparison with the authentic compound, where the M+ ion was found at m/z 254 (Mr of HQ, bis-trimethylsilylated). The retention time of compound eluting in peak 3 corresponded to that of (oxidized) benzoquinone, which eluted slightly later (6). The GC-MS analysis of this bis-trimethylsilylated fraction yielded a mass spectrum identical to that of HQ due to the displacement caused by the derivatization step.


Figure 3
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  		FIG. 3. Proposed degradation pathway of BPA in Sphingomonas sp. strain TTNP3. Type II ipso substitution and side reactions. Main pathway: one atom of molecular oxygen is introduced by the monooxygenase exclusively in the presence of NADPH and FAD as cofactors in the BPA (compound 1). The result from the ipso-hydroxylation at position C-4 is the production of a quinol intermediate (compound 2). Consequent to the rearomatization of this intermediate, the C-C bond between the semiquinol and the isopropylphenol moieties is cleaved. HQ (compound 3) is formed and further degraded into organic acids (15). The carbocationic isopropylphenol (compound 4) reacts with water to form 4-(2-hydroxypropan-2-yl)phenol (compound 5). Side-reactions: 4-isopropenylphenol (compound 6) and 4-isopropylphenol (compound 7) are formed from the carbocationic intermediate via the loss of H+ and the addition H–, respectively. A metabolite tentatively identified as 1-[2-(4-hydroxyphenyl)propan-2-yl]cyclohex-2-ene-1,4-diol (compound 9) or 2-[2-(4-hydroxyphenyl)propan-2-yl]cyclohex-2-ene-1,4-diol (compound 8) may arise from direct successive hydrogenations of the ring of the quinol intermediate or from a NIH shift with subsequent hydrogenations of the hydroxylated ring.


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  		TABLE 1. Mass spectral characteristics of bisphenol A and its metabolites

Metabolites eluting shortly after the injection peak (Fig. 2, peak 1) were not analyzed further. They were assumed to be degradation products of HQ. Previous studies of NP degradation have established that this peak contains a mixture of chromatographically unresolved polar compounds including succinate and 3,4-dihydroxybutanedioic acid (5, 6).

Identification of 4-(2-hydroxypropan-2-yl)phenol.
Analysis of the collected fraction (Fig. 2, peak 4) by LC-MS/radiodetection displayed a compound with a pseudomolecular ion at m/z 151 in the negative mode (Table 1). By analogy to the ipso substitution reaction leading to the formation of HQ and nonanol in the case of NP, the hydroxylated form of the tert-butylphenol, which is substituted as a consequence of ipso-hydroxylation of BPA, would be formed. This compound should be 4-(2-hydroxypropan-2-yl)phenol (Fig. 3, compound 5, Mr of 152). This product was further analyzed by GC-MS after derivatization, i.e., bis-trimethylsilylation (Table 1). A compound with molecular ion at m/z 296 was detected and was interpreted as the bis-trimethylsilylated derivative of 4-(2-hydroxypropan-2-yl)phenol. Contrary to GC-MS analysis of nonanol, which does not allow for the detection of the molecular ion (10), the positive charge resulting from electron ionization of bis-O-trimethylsilylated 4-(2-hydroxypropan-2-yl)phenol can be stabilized by the phenolic moiety and, thus, allows detection of the molecule peak. The ion at m/z 281 corresponded to the loss of a methyl group, while the ion at m/z 193 could be interpreted as the loss of both the trimethylsilyloxy and the methyl groups. The ion at m/z 73 corresponds to the trimethylsilyl fragment.

Identification of 4-isopropenylphenol.
In addition to the presence of remaining BPA, the LC-MS analysis of the fraction of peak 6 (Fig. 2) showed a pseudomolecular ion with a mass of 133 (Table 1). The compound was assumed to be 4-isopropenylphenol (Fig. 3, compound 6; molecular weight, 134).

Further GC-MS analysis of the trimethylsilylated compound showed evidence of a compound with a molecule ion at m/z 206 (Table 1). The molecule ion of this compound was consistent with that of the trimethylsilylated derivate of 4-isopropenylphenol (Mr, 134 + 72). Furthermore, analysis of the underivatized compound led to a mass spectrum identical to that reported for 4-isopropenylphenol (17).

Identification of 4-isopropylphenol.
The fraction corresponding to peak 7 (Fig. 2) was analyzed by LC-MS/radiodetection and led to the detection of one compound with a pseudomolecular ion displaying a mass of 135 Da (Table 1). This compound was assumed to be the deprotonated form of 4-isopropylphenol (Fig. 3, compound 7; Mr, 136,).

Further GC-MS analysis of the derivatized compound showed evidence of a metabolite characterized by a molecule ion at m/z 208 (Table 1). This mass of 208 Da was consistent with the mass expected for the O-trimethylsilylated derivative of isopropylphenol (Mr, 136 + 72). By comparative analysis with the trimethylsilylated authentic compound, this metabolite was definitely identified as 4-isopropylphenol. Furthermore, the analysis of the underivatized compound led to a mass spectrum identical to that reported for 4-isopropylphenol (17).

Other metabolites.
In the peak 5 fraction, a compound corresponding to a pseudomolecular ion at m/z 247 was detected (Table 1). Examination of the corresponding product ion spectrum (LC-MS/MS) showed the production of a stable fragment at m/z 133, corresponding to an isopropene phenolate moiety (see Fig. S1 in the supplemental material). On the basis of this mass spectrum and a similar metabolite detected in the case of an NP isomer, this metabolite was tentatively identified as 1-(2-(4-hydroxyphenyl)propan-2-yl)cyclohex-2-ene-1,4-diol (Fig. 3, compound 9; Mr, 248) and/or 2-(2-(4-hydroxyphenyl)propan-2-yl)cyclohex-2-ene-1,4-diol (Fig. 3, compound 8; Mr, 248).

Monooxygenase activity in cell extracts.
The enzymatic activity responsible for the formation of the metabolites of NP and BPA was screened in cell extracts of Sphingomonas sp. strain TTNP3. Both the low stability of the enzymatic activity at 4°C (half-life of <24 h, data not shown) and the requirements for high concentrations of cofactors made detection of the activity difficult. The conversion of 14C-labeled substrates was adequately supported by the addition of NADPH (20 mM) and FAD (1 mM) (Table 2), and the degradation products of NP were HQ and the short-chain organic acids, while the use of BPA as a substrate led to the production of the newly identified metabolites.


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  		TABLE 2. Monooxygenase activity in acellular extracts of Sphingomonas sp. strain TTNP3 in the presence of various substrates and cofactors

The use of lower concentrations of one of the cofactors caused a decrease of activity, while the omission of either led to an absence of any detectable transformation of NP and BPA. The substitution of NADPH by NADH or of FAD by FMN was quite ineffective for both substrates, which remained almost entirely intact. Further assays were carried out with 20 mM NADPH and 1 mM FAD.

Because all ipso substitution-catalyzing enzymes reported until now are P450s, assays were carried out in the presence of some typical inhibitors of cytochrome P450 (2, 27, 20). Assays carried out with octylamine and proadifene (SKF-525A) inhibited the degradation of BPA by >99% (0.1% standard deviation [SD] for n = 3) and 86.3% (SD, 6.4%), respectively, in comparison to control experiments carried out without inhibitors under the same conditions (defined as 100% activity). Octylamine and SKF-525A were also strongly effective in blocking the degradation of p353NP to 92.9% (SD, 2.0%) and 92.8% (SD, 1.7%). Another general P450 inhibitor, metyrapone, also had a slight effect on the degradation of both BPA and NP, inhibiting their conversion by 15.4% (SD, 5.3%) and 25.2% (SD, 0.8%), respectively.

Resting cells and enzymatic assays under an 18O2 atmosphere.
In order to assess the origin of the hydroxyl group introduced at the C-1 position of these xenobiotics, preliminary experiments were carried out using resting cell suspensions of Sphingomonas sp. strain TTNP3 in the presence of (nonradioactive) NP under an 18O2 atmosphere. After incubation, BSTFA-derivatized extracts of the supernatant were analyzed. The mass of the formed HQ (bis-trimethylsilylated) was 256 Da, a shift of two additional atomic mass units (amu) in comparison to that of controls performed under a normal atmosphere (16O2). The efficiency of incorporation of one atom of heavy molecular oxygen was evident. The ratio of the peaks at m/z 256 (assays with 18O2) and a peak at m/z 254 (assays with 16O2) showed that under 18O2 conditions, the stable isotope was incorporated to >95% of the formed HQ (see Table SI in the supplemental material). Furthermore, the main ion at m/z 239 resulting from the cleavage of a methyl group from one trimethylsilyl moiety of twice trimethylsilylated HQ was shifted to m/z 241. The same shift was observed for the ion at m/z 223, which was 2 amu heavier when the cells were incubated with 18O2. In order to assess whether a second atom of molecular oxygen is incorporated in the leaving group, the mass spectra of 3,5-dimethyl-3-heptanol (nonanol) were examined. No noticeable difference in the mass spectra of both 3,5-dimethyl-3-heptanols could be observed when cells were incubated under 16O2 or 18O2 atmosphere (see Table SI in the supplemental material). Because GC-MS analysis does not allow for the detection of the molecular ion of nonanol (10), the highest ion, i.e., m/z 129 (loss of a methyl) was examined in both extracts (which were not derivatized in order to avoid the loss of the very volatile nonanol). Examination of the mass spectrum of 3,5-dimethyl-3-heptanol formed during assays where NP was supplied as the substrate revealed that no molecular oxygen was incorporated. The main ion at m/z 129 (loss of a methyl group from the nonderivatized 3,5-dimethyl-3-heptanol) was present in both cases.

In order to demonstrate that molecular oxygen is also incorporated to BPA under conditions favorable to the monooxygenase activity, experiments were repeated during enzymatic assays using BPA as the substrate under an 18O2 atmosphere. The same assays were carried out in parallel with NP under an 18O2 or an 16O2 atmosphere. 18O2 was efficiently incorporated into the HQ formed from NP and BPA (see Table SI in the supplemental material). The same shift of 2 amu was also observed for NP. The nonanol and the bis-trimethylsilylated derivative of 4-(2-hydroxypropan-2-yl)phenol formed during enzymatic assays were not detectable under the experimental conditions used.

Degradation and enzymatic assays with an NP mutant.
Of the three Tn5 mutants that lost the capacity to grow on solid medium in the presence of NP as the sole carbon source, one mutant was able to grow in the presence of HQ. This mutant was tested for its ipso substitution activity. A suspension of resting cells of this mutant did not lead to the formation of HQ when incubated with 14C-labeled NP or BPA. The possibility that the degradation capacity resulted from a deficiency in a transport mechanism could also be precluded. Monooxygenase activity was assayed using cell extracts of this mutant under the optimal enzymatic assay conditions. No trace of degradation products of 14C-labeled BPA or p353NP was detected by HPLC/radiodetection and GC-MS analyses. These findings corroborate the hypothesis that the ipso substitution mechanism involved in the degradation of both BPA and NP is catalyzed by the same enzymatic system in Sphingomonas sp. strain TTNP3.


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DISCUSSION
 
Insights into the metabolism of NP by Sphingomonas strains have made clear that the ipso substitution mechanism is not restricted to halogen-substituted phenols (5, 6, 14). These bacteria are able to break highly stable C-C bonds between an aromatic carbon and the quaternary {alpha}-carbon of alkylphenol. Studies of NP have indicated the need for a structural requirement for such a reaction, i.e., the presence of a quaternary {alpha}-carbon. NP isomers (alkyl chain isomers) containing tertiary or secondary carbons at the {alpha} position resist complete degradation (11, 13). We presently found a novel degradation pathway for BPA, which starts with ring hydroxylation. Many degradation intermediates were detected and identified. The formation of HQ (Fig. 3, compound 3) constitutes the first evidence that BPA degradation by Sphingomonas sp. strain TTNP3 also involves a type II ipso substitution mechanism. Subsequent to ipso-hydroxylation, the alkyl substitutent leaves the quinol intermediate forming HQ and a carbocationic intermediate (Fig. 3, compound 4). Studies of the degradation of NP have shown that this compound is degraded to CO2 and that HQ is the intermediate used as the sole source of carbon for growth by Sphingomonas sp. strain TTNP3 (6, 10). Further degradation compounds that support the hypothesis of a type II mechanism were identified. By analogy to the degradation of NP, where the carbocationic intermediate (Fig. 3, compound 4) is further solvolyzed into nonanol, the homologue, 4-(2-hydroxypropan-2-yl)phenol (Fig. 3, compound 5), was identified as a metabolite of BPA. As for nonanol, the formation of this compound can be rationalized by the solvolysis of the carbocationic leaving moiety. Further side products, i.e., 4-isopropylphenol (Fig. 3, compound 7) and 4-isopropenylphenol (Fig. 3, compound 6) detected during experiments with resting cells are presumed to be formed by the addition of H– and loss of H+, respectively, on leaving isopropylphenolic cation. It is yet not clear whether such reactions are nonenzymatic. Interestingly, 4-isopropylphenol and 4-isopropenylphenol were detected when BPA was incubated with fungal manganese peroxidase and laccase (17, 24). In these cases, ipso attacks by these lignin-degrading enzymes cannot be excluded.

A further metabolite possibly corresponding to 1-(2-(4-hydroxyphenyl)propan-2-yl)cyclohex-2-ene-1,4-diol was detected. The signal at m/z 133 in the mass spectrum supports the hypothesis that an intact isopropyl moiety is present on this compound. The formation of 1-(2-(4-hydroxyphenyl)propan-2-yl)-cyclohex-2-ene-1,4-diol (Fig. 3, compound 9) could be explained by the fact that the quinol derivative undergoes successive hydrogenations. The keto form of analogue products, i.e., 4-hydroxy-4-(1-methyl-octyl)-cyclohex-2-enone and 4-hydroxy-4-nonyl-cyclohex-2-enone was detected during the degradation of 4-(1-methyl-octyl)-phenol and of 4-n-NP in Sphingomonas xenophaga strain Bayram (14). Nonetheless, this tentatively identified product could also correspond to 2-(2-(4-hydroxyphenyl)propan-2-yl)cyclohex-2-ene-1,4-diol (Fig. 3, compound 8). In fact, this compound could be rationalized by an NIH shift mechanism, namely the migration of the substituent to the adjacent C atom consecutively with hydroxylation, followed by hydrogenation reactions. By analogy with studies with NP (8, 14), a carbanionic shift of the isopropylphenol moiety to the adjacent carbon could occur. Another possibility is that the result of this NIH shift does not involve an intramolecular migration mechanism but rather a condensation reaction between the leaving carbocationic intermediate and the newly formed HQ (6). Such a mechanism would solve the inconsistency of the involvement of the thermodynamically improbable tertiary carbanion. In contrast to studies carried out with NP (6, 14), neither alkylbenzenediol nor alkyloxyphenol derivatives could be detected. A possible explanation is the stability of the isopropylphenol carbocation, which does not react immediately at an intramolecular level.

While the addition of a hydroxy radical at the ipso position of p-cresol can be easily achieved by chemical reactions (1) and single halogen atoms are effectively detached from phenols in biological systems (12, 27, 30), the biochemistry of ipso substitution reaction in the cases of NP and BPA is more complex. The steric hindrance at the fourth carbon position, where the hydroxyl group is introduced, outlines the fascinating aspect of this enzymatic reaction. We demonstrated that the ipso substitutions leading to alkyl-dearylation of two different para-substituted phenols, i.e., BPA and NP, containing a quaternary {alpha}-carbon were catalyzed by the same enzymatic system.

First, we showed that the conversion of both substrates was preferentially catalyzed in the presence of NADPH and FAD. Our results indicated that this enzymatic activity has quite high requirements concerning these cofactors. Nevertheless, the possibility that NADPH-oxidase acts as a "contamination" of the cell extract cannot be excluded. The experimental protocol used for the preparation of cell extract probably did not lead to the entire removal of membrane debris, which often contain high amounts of NADPH oxidase.

Second, we tested the effects of a set of specific inhibitors of P450 monooxygenase activity because a literature overview of ipso substitution reactions points to the usual involvement of P450 monooxygenase activity (12, 15, 16, 25-27). Furthermore, the degradation of BPA via the production of the rearranged product 1,2-bis(4-hydroxyphenyl)-2-propanol in Sphingomonas bisphenolicum (formerly AO1) has been demonstrated to be catalyzed by a ferredoxin reductase-ferredoxin-P450 system. We observed that octylamine, proadifene, and metyrapone (2, 20, 27) inhibited the conversion of both BPA and NP to a similar extent. No evidence concerning the involvement of a P450 system was obtained from carbon monoxide difference spectra, but the crude system may not be concentrated enough to reach a definite conclusion.

Third, we investigated the origin of the hydroxyl group which is introduced at position C-4. Using a resting cell suspension incubated with NP under an 18O2 atmosphere, we could demonstrate that molecular oxygen is present in HQ. Furthermore, examination of the mass spectra of nonanol clearly showed that the alcohol function remained unlabeled. This fact confirmed our previous hypothesis that the hydroxyl group introduced into the highly reactive leaving alkyl chain intermediate originates from a water molecule (6). Monooxygenase assays under an 18O2 atmosphere in the presence of BPA or NP led to the conclusion that both substrates are degraded by the same monooxygenation mechanism. Although no experiments with H218O were carried out, we assume that in the case of BPA, 4-(2-hydroxypropan-2-yl)phenol results from the reaction of the carbocationic intermediate with one water molecule.

Further evidence that both NP and BPA are degraded by the same enzymatic system was provided with a mutant of Sphingomonas sp. strain TTNP3, which is defective for the transformation of NP via ipso substitution reaction. We demonstrated that the initial step of both BPA and NP degradation was absent in the Tn5 mutant.

Considering the substitution pattern of the neighboring {alpha}-C and the steric hindrance at C-4 in BPA and NP, this monooxygenation reaction is unusual. The better comprehension of ipso substitution involving the scission of large groups also opens new perspectives in chemistry. For instance, ipso substitution can be used to control regioselectivity during the selective preparation of diverse substituted arenes (41). Furthermore, hydroxyl radical-mediated chemical breakdown of the herbicide 2,4-dichlorophenoxyacetic acid into 2,4-dichlorophenol occurs via an ipso substitution mechanism (29). The ipso pathway is of much interest because many xenobiotics have phenolic residues or can be readily be converted by P450s to substituted phenols, which in turn give rise to toxic hydroquinone or p-benzoquinone (27, 38). The degradation of diverse xenobiotics such as (poly)halogenated phenols and steroid hormone processes via ipso attacks (15, 16, 26, 38). Furthermore, ipso attacks lead to the formation of quinol intermediates, and the quinol formation from estrogens might be linked to estrogen-induced carcinogenicity (25).


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ACKNOWLEDGMENTS
 
We thank Willy Verstraete (LabMet, University Ghent, Belgium) for providing Sphingomonas sp. strain TTNP3.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland. Phone: 41 61 467 4344. Fax: 41 61 467 4290. E-mail: philippe.corvini{at}fhnw.de Back

{triangledown} Published ahead of print on 8 June 2007. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, August 2007, p. 4776-4784, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00329-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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