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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 K m and V max values of 9.6 M and 6.8 mol min ؊1 mg of protein ؊1 , respectively.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 METHODSOrganism 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) NH 4 NO 3 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. 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 ϫ 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 Ϫ1 . 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) ...