Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134

Abstract

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

FIG. 1

Conversion of 3NP (A) and 2C5NP (B) to the corresponding hydroxylamino derivatives by 3NP nitroreductase. (A) 3NP (0.51 mM), NADPH (2.7 mM), and partially purified 3NP nitroreductase (63 mU/ml [44 ng/ml of protein]) were incubated in 50 mM phosphate buffer (pH 7.5). (B) 2C5NP (0.96 mM), NADPH (3 mM), NADH (3 mM), and partially purified 3NP nitroreductase (56 mU/ml [44 ng/ml of protein]) were incubated in 62 mM phosphate buffer (pH 7). Both experiments were carried out under an argon atmosphere at 30°C. Samples were analyzed by HPLC.

FIG. 2

Conversion of 2C5NP by induced resting cells of R. eutropha JMP134. 2C5NP (0.65 mM) and resting cells (A₅₄₆ = 27) were incubated in 50 mM phosphate buffer (pH 7) under an argon atmosphere at 30°C. The decrease in 2C5NP (□) and the formation of aminohydroquinone (○) and chloride (▵) were determined by HPLC.

FIG. 3

Conversion of 2C5NP by 3NP nitroreductase (step 1), 3HAP mutase (step 2), and 3NP-grown cells (step 3) of R. eutropha JMP134. At time zero, partially purified 3NP nitroreductase (0.56 U) was added to 62 mM phosphate buffer (pH 7) with 2C5NP (0.96 mM), NADPH (3 mM), and NADH (3 mM). After 61.5 min purified 3HAP mutase (0.72 U) was added, and after 170 min 3NP-grown cells (A₅₄₆ = 13) were added. Incubation was carried out at 30°C under anaerobic conditions. Concentrations of 2C5NP (□), 2-chloro-5-hydroxylaminophenol (◊), 2-amino-5-chlorohydroquinone (○), and aminohydroquinone (▵) were analyzed by HPLC. In order to estimate the concentration of 2-amino-5-chlorohydroquinone, aminohydroquinone was used as a standard.

FIG. 4

Initial steps of 3NP and 2C5NP degradation by R. eutropha JMP134.