Abstract
The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.
The 3-nitrophenol (NP)-induced enzyme pathway in Pseudomonas putida 2NP8 is a nitroreductase-initiated metabolic system (12, 13). This system converted nitrobenzene (NB), a cometabolic substrate, into aminophenol, with subsequent hydrolysis into ammonia and benzoquinone (Fig. 1A; R=H). We postulated that this strain converted the growth substrate 3-NP into hydroxylquinol via oxidation of aminohydroquinone into imine, hydrolysis of the imine into quinone, and reduction of the quinone (13). Many reports describe hydroxylquinol as an intermediate in bacterial metabolism of a wide range of aromatic compounds (4, 8, 10). Here we describe the substrate selectivity of the 3-NP-induced metabolic system.
FIG. 1.
Proposed pathway for ammonia-release from NAs and transformation of nitrobenzyl alcohol and nitrobenzaldehyde in P. putida 2NP8. R indicates one or two substitutents; bracket indicates unidentified compound; X indicates that reaction did not occur.
Transformation of NAs by 3-NP-grown cells.
We used a previously reported procedure (11–13) to test transformation of 30 nitroaromatic compounds (NAs) by 3-NP-grown cells of P. putida 2NP8. Initial transformation rates (Table 1) of most of the NAs were close to the transformation rates of 3-NP and NB. Loss of substrates in controls with killed cells was negligible for all NAs and 2-nitrofuran, except for 1-nitronaphthalene, 50% of which was retained by biomass within a 3-h incubation period.
TABLE 1.
Biotransformation of NAs by 3-NP-grown cells of P. putida 2NP8 and ammonia production
| Nitroaromatic substrate | Substrate initially added (mmol) | Transformation ratea (102 μmol/ h/mg dcw) | Ammonia produced (mmol, 3 h) |
|---|---|---|---|
| 3-NP | 1.4 | 8.8 | 1.3 |
| 4-Chloro-3-NP | 1.2 | 7.2 | 1.2 |
| 2-NP | 1.4 | 5.6 | 0.4 |
| 4-Chloro-2-NP | 1.2 | 5.2 | 1.2 |
| 4-NP | 1.4 | 2.4 | 0 |
| 2,4-DNP | 1.1 | 0.4 | 0 |
| 2,4,6-Trinitrophenol | 0.87 | 0.08 | 0 |
| 4-Nitrocatechol | 1.3 | 4.8 | 0.6 |
| 3-Nitroaniline | 1.4 | 4 | 0 |
| Nitrobenzene | 1.6 | 10 | 1.4 |
| 4-Chloronitrobenzene | 1.2 | 7.6 | 1.2 |
| 3-Chloronitrobenzene | 1.2 | 7.6 | 1.1 |
| 2-Nitrotoluene | 1.4 | 8.4 | 1.4 |
| 4-Nitrotoluene | 1.4 | 8.8 | 1.3 |
| 2-Nitroanisole | 1.3 | 7.2 | 1 |
| 3-Nitroanisole | 1.3 | 8 | 0.95 |
| 2-Nitrobenzoate | 1.2 | 0.4 | 0 |
| 3-Nitrobenzoate | 1.2 | 6.8 | 1.32 |
| 4-Nitrobenzoate | 1.2 | 6.8 | 1.01 |
| 2-Nitrobenzylalcohol | 1.3 | 8 | 0.95 |
| 3-Nitrobenzylalcohol | 1.3 | 8 | 1.01 |
| 4-Nitrobenzylalcohol | 1.3 | 8 | 1 |
| 2-Nitrobenzaldehyde | 1.3 | 8 | 0.06 |
| 4-Nitrobenzaldehyde | 1.3 | 8 | 1.1 |
| 3-Nitrobenzonitrile | 1.3 | 8 | 0.97 |
| 2,4-Dinitrotoluene | 1.1 | 6.8 | 0.93 |
| 3,4-Dinitrotoluene | 1.1 | 6.4 | 0.62 |
| 2,6-Dinitrotoluene | 1.1 | 6.8 | 1.2 |
| 2-Nitrofuran | 1.8 | 1.6 | 0 |
| 1-Nitronaphthalene | 1.2 | 6.8 | 0.69 |
Calculation of transformation rate was based on substrate disappearance within 1.5 h of incubation. Percent substrate transformation (3 h) >99% unless otherwise indicated; 2-NP, 83%; 4-nitrocatechol, 76%; 4-NP, 46%; 2,4-DNP, 8.3%; picric acid, 0; 2-nitrofuran, 14%; 3-nitroaniline, 51%. dcw, dry cell weight.
Transformation rates were greatly affected by hydroxyl groups located at the 2- or 4-position relative to the nitro group or by a carboxylic group at the 2-position only. Cells showed good transformation ability toward all of the position-4-substituted NAs, including dinitrotoluenes, except where that substituent was a hydroxyl group. The cells also quickly transformed all of the NAs with substitutions, other than hydroxyl or carboxyl groups, at the 2-position relative to the nitro group. The presence of a hydroxyl group at the 2- and 4-position relative to the nitro group reduced the transformation rate. We observed low or no transformation activity toward 4-NP, 2,4-dinitrophenol, 2,4,6-trinitrophenol, and 2-nitrobenzoic acid. 2-NP had a lower transformation rate than 3-NP. Substitutions at the 3-position did not reduce the transformation rate.
It was noteworthy that 1-nitronaphthalene was also quickly removed. 2-Nitrofuran was transformed at a relatively low rate, indicating that the initial nitroreductase of this 3-NP-induced enzyme system (12) may be different from the nonspecific nitrofuran nitroreductase found in Escherichia coli (1, 2).
Uninduced cells transformed only nitrobenzyl alcohol and nitrobenzaldehyde.
Glucose-grown cells (8 mg of wet cells/ml [1.5 h]) exhibited little biotransformation activity toward all of the 30 NAs except for 2- or 4-nitrobenzaldehyde and 3- or 4-nitrobenzyl alcohol, which were transformed solely through oxidation and reduction of the aldehyde or alcohol group (Fig. 1B through D). Alcohol yields from 2- and 4-nitrobenzaldehyde were 40 and 0.2%, respectively. Acid yields from the latter substrates were 60 and 92%, respectively. Acid yields from 3- and 4-nitrobenzyl alcohol were 14 and 4%, respectively. The ammonia-producing activity observed in the 3-NP-grown cells was absent in glucose-grown cells.
3-NP-grown cells transformed NAs into ammonia.
Ammonia release from many of the substrates transformed by 3-NP-grown cells appeared to be stoichiometric (Table 1). Lower ammonia production was observed from 2-NP, 4-nitrocatechol, and 3,4-dinitrotoluene. No nitrite was detected (9) in the 3-NP-grown cell transformation tests. Thus, the broad substrate specificity of this enzyme system was not limited to the initial nitro group reduction but extended further down to ammonia production.
The initial enzyme of the 3-NP metabolic systems in P. putida B2 (5) and Ralstonia eutropha JMP134 (7) is also nitroreductase, which has broad substrate specificity. Intact cells of P. putida B2 pregrown on 3-NP or its cell-free extract did not convert the NAs other than 3-NP into ammonia (5). 3-NP-grown cells of R. eutropha JMP134 converted 3-NP and 2-chloro-5-NP into ammonia but converted NB into dead-end amino phenols (7). The enzyme system in P. putida 2NP8 and R. eutropha JMP134 (7) did not attack 4-NP, 2,4-dinitrophenol, and 2-nitrobenzoate. P. putida B2 exhibited a low capacity to reduce 4-NP and 2-nitrobenzoate (5). R. eutropha JMP134 reduced picric acid but not 2-nitrotoluene (7). In contrast, P. putida 2NP8 transformed 2-nitrotoluene but not picric acid.
Metabolites from transformation of NAs by the 3-NP-grown cells.
P. putida 2NP8 cometabolized NB into ammonia and produced nitrosobenzene, hydroxylaminobenzene, aminophenol, N-acetylaminophenol, benzoquinone and hydroquinone, and catechol (12, 13). Initial rates for transformation of most NAs were close to those observed for 3-NP and NB with stoichiometric production of ammonia, suggesting that the same pathway was followed.
The reversed-phase C-18 column used to analyze metabolites retains hydrophobic compounds more strongly than hydrophilic compounds. Biotransformation of NAs by 3-NP-grown cells produced unique metabolites with strong UV absorbance at 254 nm. Metabolites from the transformation of NB were separated into two groups: those that were more hydrophobic than the substrate, such as the nitroso compound (retention time [Rt], 8.1 min) and those more hydrophilic than the substrate, such as hydroxylaminobenzene, aminophenol, and quinone (Rt, 2.8 to 3.3 min) produced by further transformation of the nitroso compound. We observed an apparently similar metabolite formation sequence in transformation of other substrates (Table 2). Metabolite formation from substrates such as 4-Cl-3-NP was low, which may be due to more rapid transformation of the metabolites. Biotransformation time-courses of all utilized substrates demonstrated that substrate removal was accompanied by formation of metabolites and ammonia (Fig. 2A through F).
TABLE 2.
HPLC Rt of metabolites formed from NAs when incubated with 3-NP-grown cells of P. putida 2NP8a
| Nitroaromatic substrate | HPLC Rt of substrate and metabolite (min)
|
||
|---|---|---|---|
| Rt of substrate (Rt[s]) | Rt of metabolite (Rt[m])
|
||
| Group I metaboliteb Rt(m) < Rt(s) | Group II metabolitec Rt(m) > Rt(s) | ||
| 3-NP | 5.5 | − | − |
| 4-Chloro-3-NP | 8.4 | − | 16.3 |
| 2-NP | 7.0 | 3.5 | 7.5, 7.9 |
| 4-Chloro-2-NP | 12.3 | 5.2, 8.1 | − |
| 4-Nitrocatechol | 4.1 | − | − |
| Nitrobenzene | 7.0 | 2.8, 3.3 | 8.1, 10 |
| 4-Chloronitrobenzene | 11.8 | 5.4, 7.1, 10.2 | − |
| 3-Chloronitrobenzene | 13.9 | 3.3, 3.5, 5.4 | − |
| 2-Nitrotoluene | 10.3 | 3.8 | − |
| 4-Nitrotoluene | 11.0 | 5.2, 10.2 | − |
| 2-Nitroanisole | 5.7 | 3.2 | 10.5 |
| 3-Nitroanisole | 10.1 | 3.5, 8.5 | − |
| 3-Nitrobenzoic acid | 5.9 | − | 6.6, 7.1 |
| 4-Nitrobenzoic acid | 6.3 | 2.9 | 7.3 |
| 2-Nitrobenzyl alcohol | 4.51 | 2.7, 3.0, 3.9 | 6.8, 7.4 |
| 3-Nitrobenzyl alcohol | 4.47 | 2.6, 3.0, 3.5 | 7.0, 7.6 |
| 4-Nitrobenzyl alcohol | 4.45 | 2.9, 4.1 | 6.9, 7.4, 11.8 |
| 2-Nitrobenaldehyde | 4.8 | 3.9 | − |
| 4-Nitrobenzaldehyde | 5.2 | 2.9 | 6.4, 6.9, 7.5, 20.4, 22.0 |
| 3-Nitrobenzonitrile | 4.9 | − | 10.0 |
| 2,4-Dinitrotoluene | 9.4 | 3.2, 3.7 | 11.8, 22.1 |
| 3,4-Dinitrotoluene | 7.3 | 3.8 | 8.8, 9.3 |
| 2,6-Dinitrotoluene | 9.07 | 3.7 | 13.3 |
| 1-Nitronaphthalene | 19.7 | 7.1 | 22.2 |
Column conditions: Zorbax SB-C 18 column; solvent, 60% aqueous methanol (0.1% trifluoroacetic acid); flow rate, 1 ml/min; UV wavelength, 254 nm. Biotransformation time, 3 h. −, no peak appeared in this Rt region.
Peaks with Rt smaller than those of the substrate indicate a hydroxylamino compound, aminophenol, N-acetylaminophenol, quinones, and catechol compounds, which are more hydrophilic than the substrate. Multiple values mean that multiple metabolites were observed.
Peaks with Rt larger than those of substrate indicate nitroso or condensed products (dimer), which are more hydrophobic than substrate. Rt order of metabolites: Rt(dimer) > Rt(nitroso compound) > Rt(NAs) > Rt(hydroxylamino compound) > Rt(N-acetylaminophenol) ∼ Rt(catechol) ∼ Rt(aminophenol) ∼ Rt(quinone) (13). Multiple values mean that multiple metabolites were observed.
FIG. 2.
Times courses of biotransformation of NAs by resting cells of P. putida 2NP8 grown on 3-NP. Biotransformation conditions: 4 ml of sodium phosphate buffer (25 mM, pH 7.3), aerobic, 150 rpm shaker, 26°C, 8 mg of wet cells. High-pressure liquid chromatography (HPLC) analytical conditions: Zorbax SB-C 18 column; solvent, 60% aqueous methanol solution (containing 0.1% trifluoroacetic acid); flow rate, 1 ml/min; UV detector wavelength, 254 nm. (A) 4C12NP, 4-chloro-2-nitrophenol; (B) 3C1NB, 3-chloronitrobenzene; (C) 1NNT, 1-nitronaphthalene; (D) 4NBAL, 4-nitrobenzyl alcohol; (E) 4NBAD, 4-nitrobenzaldehyde; (F) 4NBA, 4-nitrobenzoic acid. m5.2, metabolite with HPLC retention time of 5.2 min.
Biotransformation of nitrobenzyl alcohol and nitrobenzaldehyde into ammonia.
3-NP-grown cells transformed 3- or 4-nitrobenzyl alcohol with stoichiometric production of ammonia. However, low amounts of nitrobenzoic acids were observed (Fig. 2D), which are also substrates of the 3-NP-induced enzymes. Therefore, conversions of the nitrobenzyl alcohols to ammonia via the acid intermediates may also have occurred.
Transformation of 2-nitrobenzaldehyde by 3-NP-grown cells produced little ammonia but a significant amount of 2-nitrobenzoic acid (yield, 50%). Ammonia production from 4-nitrobenzaldehyde was almost stoichiometric, while yield of 4-nitrobenzoic acid was only 3.5% (Fig. 2E). Production of acid was likely the result of the constitutive aldehyde-oxidizing activity as shown in the glucose-grown cells (Fig. 1D). Reduction of the aldehyde to alcohol, as observed in the glucose-grown cells (Fig. 1B and D), might also occur in the 3-NP-grown cells. Thus possible routes for ammonia release from 4-nitrobenzaldehyde involve either direct transformation with the aldehyde group intact or transformation after it was converted to 4-nitrobenzoic acid or 4-nitrobenzyl alcohol.
Two Pseudomonas sp. strains (3, 6), with 4-nitrotoluene as growth substrate, transformed 4-nitrotoluene to 4-nitrobenzyl alcohol, 4-nitrobenzaldehyde, and 4-nitrobenzoic acid. These strains metabolized 4-nitrobenzoic acid to ammonia using nitroreductases. Our strain P. putida 2NP8 did not oxidize 4-nitrotoluene.
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