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) ...

Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.

We aimed to examine the effects of ingestion of different amounts of carbohydrate (CHO) after endurance exercise on neutrophil count, circulating cytokine levels, and the markers of neutrophil activation and muscle damage. Nine participants completed three separate experimental trials consisting of 1 h of cycling exercise at 70% V·O2 max, followed by ingestion of 1.2 g CHO·kg body mass−1·h−1 (HCHO trial), 0.2 g CHO·kg body mass−1·h−1 (LCHO trial), or placebo (PLA trial) during the 2 h recovery phase in random order. Circulating glucose, insulin, and cytokine levels, blood cell counts, and the markers of neutrophil activation and muscle damage were measured. The concentrations of plasma glucose and serum insulin at 1 h after exercise were higher in the HCHO trial than in the LCHO and PLA trials. Although there were significant main effects of time on several variables, including neutrophil count, cytokine levels, and the markers of neutrophil activation and muscle damage, significant time × trial interactions were not observed for any variables. These results suggest that CHO ingestion after endurance exercise does not enhance exercise-induced increase in circulating neutrophil and cytokine levels and markers of neutrophil activation and muscle damage, regardless of the amount of CHO ingested.