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Applied and Environmental Microbiology, September 2003, p . 5410-5413, Vol . 69, No . 9

The Metabolic Pathway of 4-Aminophenol in Burkholderia sp . Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents

Shinji Takenaka,1 Susumu Okugawa,2 Maho Kadowaki,1 Shuichiro Murakami,1 and Kenji Aoki1*

Department of Biofunctional Chemistry, Faculty of Agriculture,1 Division of Science of Biological Resources, Graduate School of Science and Technology, Kobe University, Rokko, Kobe, Japan2

Received 10 March 2003/ Accepted 2 July 2003


   ABSTRACT

 
Burkholderia sp . strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source . A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis . Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol . 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid . The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with Km and Vmax values of 9.6 µM and 6.8 µmol min-1 mg of protein-1, respectively .


   INTRODUCTION

 
4-Aminophenol has highly toxic and mutagenic effects and induces DNA cleavage in mouse and human lymphoma cells (12, 22) . This compound is an intermediate in the degradation of hydroxyacetanilide (7) and azo dyes (19) . However, little is known about the metabolism of 4-aminophenol by bacteria (1) . 3-Nitrophenol-grown cells of Ralstonia eutropha JMP 134 convert nitrobenzene to hydroxylaminobenzene, 2-aminophenol, and 4-aminophenol (16) . Hydroxylaminobenzene is transformed by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8 to 1,4-benzenediol via 4-aminophenol (25) . A number of reports indicate that 4-aminophenol might be a key intermediate in the biodegradation of nitrobenzenes and amines (7, 19, 25) . Our aim was to elucidate a biodegradation pathway for 4-aminophenol by analyzing metabolites .

Here we report the isolation of a 4-aminophenol-assimilating bacterium and propose a metabolic pathway for 4-aminophenol . In addition, the characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from strain AK-5 is described .


   MATERIALS AND METHODS

 
Organism and growth conditions.
Strain AK-5 was enriched from rice field soil from the Hyogo Prefecture . The basal medium containing 4-aminophenol was prepared by methods described previously (3) . Succinate-glucose medium was a modified basal medium containing 1.0% (wt/vol) sodium succinate, 1.0% (wt/vol) D-glucose, and 0.04% (wt/vol) NH4NO3 as the sole carbon and nitrogen sources instead of 4-aminophenol .

Purification of 1,2,4-trihydroxybenzene 1,2-dioxygenase.
1,2,4-Trihydroxybenzene 1,2-dioxygenase activity was assayed by the method of Latus et al . (10) . The molar extinction coefficient of 4.44 x 103 at 243 nm for maleylacetic acid was used (20) . Protein concentrations were measured by the method of Lowry et al . (11) .

Cells (25 g [wet weight]) of strain AK-5 were suspended in 20 mM Tris-HCl (pH 8.0) (buffer A) . Cell extract (fraction 1) was prepared and treated with streptomycin sulfate (fraction 2) as described previously (3) . Fraction 2 was fractionated with ammonium sulfate (32 to 50% saturation) . After centrifugation (20,000 x g for 10 min), the pelleted precipitate was dissolved in buffer A . The solution was dialyzed against buffer A (fraction 3, 90 ml) . Fraction 3 was applied to a DE52 cellulose column (2.1 by 26 cm), and proteins were eluted with a linear gradient (0 to 0.4 M NaCl) at a flow rate of 40 ml h-1 . The active fractions were pooled (fraction 4; 60 ml) . Fraction 4 was applied to a DEAE-Cellulofine A-800 column (2.0 by 15 cm), and proteins were eluted with a linear gradient (0 to 0.35 M) of NaCl at a flow rate of 30 ml h-1 . The active fractions were pooled (fraction 5; 30 ml) . Fraction 5 was applied to a phenyl-Cellulofine column (1.6 by 7.5 cm), and proteins were eluted with a linear gradient (0.5 to 0 M) of (NH4)2SO4 at a flow rate of 30 ml h-1 . The active fractions were pooled (fractions 6; 28 ml) . Fraction 6 was concentrated, and the solution was loaded onto a Cellulofine GCL-1000 sf column (3.2 by 58 cm); proteins were eluted with buffer A containing 0.2 M NaCl at a flow rate of 20 ml h-1 . The enzyme purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21) .

Substrate specificity.
The benzene ring cleavage of 1,4-benzenediol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, 4-nitrocatechol, protocatechuic acid, and pyrogallol was monitored spectrophotometrically . Inhibition of the enzyme activity by substrate analogues was examined by incubating the enzyme (25 µg ml-1) with each analogue (0.05 mM) in 3 ml of 100 mM sodium-potassium phosphate buffer (pH 7.5) at 24°C for 1 min . The enzyme reaction was started by adding 1,2,4-trihydroxybenzene .

Effect of various compounds on enzyme activity.
The effect of metal salts and chelating and sulfhydryl agents on enzyme activity with 1,2,4-trihydroxybenzene as the substrate was tested by methods described previously (18) .

Production and isolation of metabolites.
The reaction mixture contained 84 ml of 100 mM sodium-potassium phosphate buffer (pH 7.5), 3.0 ml of cell suspension (0.57 mg [dry weight] of cells ml-1), and 3 ml of 10 mM 4-aminophenol . After incubation with shaking at 30°C for 5 min, cells were broken by ultrasonic disintegration . The solution was then adjusted to pH 3.0 with 3 N HCl, and precipitated proteins were removed by centrifugation at 20,000 x g for 10 min . The supernatant was concentrated with an evaporator to 20 ml and then extracted with ethyl acetate . The upper layer was recovered and evaporated to dryness . The accumulated products reacted with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) at 90°C for 1.5 h . The trimethylsilylated products were analyzed by gas chromatography (GC)-mass spectrometry (MS) as described below .

Consumption of molecular oxygen by whole cells.
Possible metabolites were assayed in oxygen uptake experiments with 4-aminophenol-grown cells by using a Clark-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, Colo.) . Cells were grown in 4-aminophenol or succinate-glucose medium . The reaction mixture contained 3.0 ml of 50 mM sodium-potassium phosphate buffer (pH 7.0) and 0.1 ml of cell suspension (0.57 mg [dry weight] of cells ml-1) . The reaction was started by adding 0.1 ml of 10 mM 4-aminophenol, 1,4-benzenediol, 1,4-benzoquinone, phenol, or catechol, and the reaction mixture was incubated at 24°C .

Identification of the reaction product (compound IV) from 1,2,4-trihydroxybenzene.
The reaction mixture contained 100 ml of 100 mM sodium-potassium phosphate buffer (pH 7.5), 4.0 ml of enzyme solution (25 µg ml-1), and 4 ml of 5 mM 1,2,4-trihydroxybenzene . After incubation at 24°C for 15 min, the mixture was concentrated to 30 ml with a rotary evaporator . The mixture was then extracted and derivatized for GC and GC-MS analysis essentially as described above .

Analytical methods.
UV absorption spectra of reaction products were recorded with a Beckman DU 650 spectrophotometer . The trimethylsilylated compounds were analyzed with a Hitachi M-2500 mass spectrometer under conditions described previously (18) . 4-Aminophenol in the growing culture was determined by a diazo coupling reaction (14) . A molar extinction coefficient of 3.4 x 103 at 578 nm for the diazotized compound was used . The apparent molecular mass of the native enzyme was determined by gel filtration on Cellulofine GCL-1000 sf . The molecular mass of the enzyme subunits was measured by SDS-PAGE (21) .

Chemicals.
4-Aminophenol, 1,2,4-trihydroxybenzene (hydroxyhydroquinone), 1,4-benzenediol (hydroquinone), catechols, and BSTFA were purchased from Wako Pure Chemicals (Osaka, Japan) . DE52 cellulose was from Whatman (Madison, Wis.), and DEAE-Cellulofine A-800, phenyl-Cellulofine, and Cellulofine GCL-1000 sf were from Seikagaku (Tokyo, Japan) .


   RESULTS AND DISCUSSION

 
Identification of a 4-aminophenol-assimilating bacterium.
Strain AK-5 is a motile rod of 0.8 to 1.2 by 2.4 to 3.6 µm with polar flagella . It is aerobic, gram negative, not spore forming, and catalase and oxidase positive . It produces acid oxidatively from D-glucose, D-fructose, D-sorbitol, D-mannitol, lactose, maltose, and sucrose . It does not produce H2S, indole, or acetoin and does not hydrolyze gelatin . The nucleotide sequence of the 16S rRNA gene of strain AK-5 (1,520 bp; accession no . AB103080) was 97.9 and 98.1% identical to that of Burkholderia sp . strain NF100 (AB025790) and Burkholderia sp . strain S4.9 (AF247496), respectively (6, 8) . Thus, strain AK-5 was identified as a species of Burkholderia .

Strain AK-5 grew well on 4-aminophenol as the sole carbon, nitrogen, and energy source (Fig . 1) . 4-Aminophenol was rapidly degraded during the exponential phase . The consumption of 4-aminophenol correlated with an increase in cell density and protein content . High concentrations of 4-aminophenol (>18.0 mM) inhibited growth . Strain AK-5 grew well at pHs from 5 to 6.5 and poorly at pHs >6.5 .


FIG . 1 . Growth of Burkholderia sp . strain AK-5 on 4-aminophenol . Strain AK-5 was grown in basal medium containing 4-aminophenol (1.2 g · liter-1) without yeast extract in a 500-ml flask at 30°C with shaking . Disappearance of 4-aminophenol ({blacksquare}; {square}, control) was measured spectrophotometrically by using a diazo coupling reaction (14) . The increase in cell density was determined by measuring the optical density at 660 nm (•) or the protein content ({triangleup}) of the culture.

 
Purification and properties of the purified dioxygenase.
The 1,2,4-trihydroxybenzene 1,2-dioxygenase from strain AK-5 was present in cell extracts of 4-aminophenol-grown cells but not in cell extracts of succinate-glucose-grown cells; therefore, the synthesis of the enzyme was inducible . The enzyme was purified 108-fold, with an overall yield of 0.9% (Table 1) . The apparent molecular mass was determined to be 85 kDa by gel filtration, and the molecular mass was determined to be 81 kDa by SDS-PAGE, which indicated that the enzyme is a monomer .


TABLE 1 . Purification of 1,2,4-trihydroxybenzene 1,2-dioxygenase from Burkholderia sp . strain AK-5

 
After DE52 chromatography, the dioxygenase from strain AK-5 was stable for several weeks in buffer A containing 250 mM NaCl . The dioxygenase from Burkholderia cepacia AC1100 is also stable at a high salt concentration (5) . The enzyme from strain AK-5 maintained more than 100% activity after a 10-min incubation at temperatures up to 50°C and showed maximal activity at pH 7.0 . The enzyme had a high activity only for 1,2,4-trihydroxybenzene, with Km and Vmax values of 9.6 µM and 6.8 µmol · min-1 · mg of protein-1, respectively . Such a remarkably narrow substrate specificity is shared with the dioxygenase from Trichosporon cutaneum (17) . Among the substrate analogues tested, 1,4-benzenediol, 4-methylcatechol, and 4-chlorocatechol decreased the enzyme activity for 1,2,4-trihydroxybenzene to 58, 66, and 25%, respectively . Among the metal salts tested, the enzyme was completely inhibited by 1 mM HgCl2, 1 mM MgSO4, and 1 mM AgNO3 . The addition of 1 mM {alpha},{alpha}'-dipyridyl, EDTA, o-phenanthroline, or NaN3 decreased the enzyme activity to 48, 49, 0, and 25%, respectively .

Proposed pathway of 4-aminophenol metabolism.
4-Aminophenol (0.20 mM) was degraded, with elimination of ammonia (0.17 mM), by 4-aminophenol-grown whole cells of strain AK-5 . Metabolites were analyzed by GC and GC-MS . Trimethylsilylated 4-aminophenol (M+ = 253) had a GC retention time of 10.7 min . Major peaks at 8.6 and 12.1 min were also observed . The mass spectra (Table 2) and the GC retention times (Rt) of compounds II and III agreed with those of the trimethylsilylated authentic 1,4-benzenediol (Rt = 8.6 min) and 1,2,4-trihydroxybenzene (Rt = 12.1 min), respectively (Fig . 2) . The enzymatic reaction product derived from 1,2,4-trihydroxybenzene showed an absorption peak at 243 nm . The mass spectrum of the trimethylsilylated reaction product (compound IV) is in agreement with that of maleylacetic acid (Table 2) (15) .


TABLE 2 . Mass spectra of the metabolites from 4-aminophenol and reaction product from 1,2,4-trihydroxybenzene

 

FIG . 2 . Proposed pathway of 4-aminophenol metabolism in Burkholderia sp . strain AK-5 and comparison to the metabolic pathways of aniline and aniline derivatives . (a) Proposed pathway of 4-aminophenol metabolism . I, 4-aminophenol; II, 1,4-benzenediol; III, 1,2,4-trihydroxybenzene; IV, maleylacetic acid . (b) Metabolic pathways of aniline and aniline derivatives in Rhodococcus erythropolis AN-13 (2), Pseudomonas putida mt-2 (12), Moraxella sp . strain G (24), and Hydrogenophaga palleronii S5 (4) . I, aniline (R = H), p-toluidine (R = CH3), 4-chloroaniline (R = Cl), 4-aminobenzoic acid (R = COOH), and 4-aminobenzenesulfonic acid (R = SO3H); II, 1-amino-2-hydrodiols; III, catechol (R = H), 4-methylcatechol (R = CH3), 4-chlorocatechol (R = Cl), protocatechuic acid (R = COOH), and 4-sulfocatechol (R = SO3H).

 
The oxygen uptake rates of 4-aminophenol-grown whole cells with 4-aminophenol, 1,4-benzenediol, and 1,2,4-trihydroxybenzene were 22, 10, and 12 µmol min-1 mg of protein-1, respectively . In contrast, the oxygen uptake rates of succinate-glucose-grown whole cells with these compounds were less than 1 µmol min-1 mg of protein-1 . The oxygen uptake rates of 4-aminophenol- and succinate-glucose-grown whole cells with phenol, catechol, or 1,4-benzoquinone were less than 1 µmol min-1 mg of protein-1 These results indicated that the enzymes responsible for 4-aminophenol, 1,4-benzenediol, and 1,2,4-trihydroxybenzene metabolism were induced in 4-aminophenol-grown cells .

Figure 2a shows the proposed metabolic pathway of 4-aminophenol in strain AK-5 . 4-Aminophenol was converted to 1,2,4-trihydroxybenzene via 1,4-benzenediol; 1,2,4-trihydroxybenzene 1,2-dioxygenase catalyzed the conversion of 1,2,4-trihydroxybenzene to maleylacetic acid . Presumably, the benzene ring of 4-aminophenol is subjected to two hydroxylation steps to yield 1,2,4-trihydroxybenzene . The proposed pathway differs from previously reported metabolic pathways for aniline and anilines with a methyl-, chloro-, sulfo-, or carboxy-functional-group substituent at the C-4 position (Fig . 2b) (2, 4, 13, 24) . The initial reaction in the degradation of anilines is catalyzed by a dioxygenase and yields the corresponding 1-amino-2-hydrodiols as the first metabolites . Subsequent oxidation of 1-amino-2-hydrodiols leads to the formation of the catechols . The dioxygenation and dehydrogenation steps to form catechols in these species are similar irrespective of which functional group at the C-4 position of aniline is the electron donor or electron acceptor .

Hughes et al . (9) have reported that, in Pseudomonas putida strain TW3, 4-hydroxylaminobenzoate lyase converts 4-hydroxylaminobenzoate to protocatechuate, replacing the amino group by a hydroxyl group . Likewise, strain AK-5 could possibly require lyase activity in the initial step of 4-aminophenol metabolism, which we propose to be the direct conversion of 4-aminophenol to hydroquinone .

When the purified enzyme was added to a reaction mixture containing 1,2,4-trihydroxybenzene, the absorption peak at 243 nm, corresponding to maleylacetic acid, and the absorption peak at 260 nm, arising from the auto-oxidation of 1,2,4-trihydroxybenzene (23), increased slowly . In contrast, the auto-oxidation product did not accumulate when cell extract and the (NH4)2SO4 fraction were used in the assay . The cell extract might contain enzymes that inhibit the nonenzymatic reaction or that reduce the product . We are currently investigating enzymes involved in the transformation of the auto-oxidation product .

 


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan . Phone: 81 78 803 5891 . Fax: 81 78 882 0481 . E-mail: kaoki{at}kobe-u.ac.jp .


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