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Applied and Environmental Microbiology, November 2000, p. 4742-4750, Vol. 66, No. 11
Department of
Bacteriology,1 Department of Civil and
Environmental Engineering,2 and
Department of Biochemistry,3 University of
Wisconsin, Madison, Wisconsin 53706
Received 8 May 2000/Accepted 11 August 2000
The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by
purified XenB, an NADPH-dependent flavoprotein oxidoreductase from
Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-14C]TNT preparations. XenB catalyzed the
reduction of TNT either by hydride addition to the aromatic ring or by
nitro group reduction, with the accumulation of various tautomers of
the protonated dihydride-Meisenheimer complex of TNT,
2-hydroxylamino-4,6-dinitrotoluene, and
4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these
metabolites were nonenzymatic and resulted in predominant formation of
at least three dimers with an anionic m/z of 376 as
determined by negative-mode electrospray ionization mass spectrometry
and the release of ~0.5 mol of nitrite per mol of TNT consumed. The
extents of the initial enzymatic reactions were similar in the
presence and in the absence of O2, but the dimerization
reaction and the release of nitrite were favored under aerobic
conditions or under anaerobic conditions in the presence of
NADP+. Reactions of chemically and enzymatically
synthesized and high-pressure liquid chromatography-purified TNT
metabolites showed that both a hydroxylamino-dinitrotoluene isomer and
a tautomer of the protonated dihydride-Meisenheimer complex of TNT were
required precursors for the dimerization and nitrite release reactions.
The m/z 376 dimers also reacted with either dansyl chloride
or N-1-naphthylethylenediamine HCl, providing evidence for
an aryl amine functional group. In combination, the experimental
results are consistent with assigning the chemical structures of the
m/z 376 species to various isomers of
amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of
these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.
Bacterial transformation of the
explosive 2,4,6-trinitrotoluene (TNT) (Table
1, compound 1) has been shown to proceed
via two different biochemical routes: direct 2e
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transformation of 2,4,6-Trinitrotoluene by Purified
Xenobiotic Reductase B from Pseudomonas fluorescens
I-C
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
reduction
of the aromatic ring via hydride addition and successive 2e reduction of the nitro groups. The extents of these
reactions and the predominant products depend on the type of
microorganism involved and the environmental conditions. Here we
consider the action of an enzyme purified from an aerobic bacterium,
Pseudomonas fluorescens I-C.
TABLE 1.
TNT metabolites produced by XenB as revealed by ESI-MS
and HPLC analyses
Direct reduction of the aromatic ring has been reported to be catalyzed by aerobic bacteria (8, 31, 32), and the products include a hydride-Meisenheimer complex (H-TNT), a dihydride-Meisenheimer complex (2H-TNT), and several protonated tautomers of 2H-TNT (2H-TNT · H+). Reduction of the nitro group in aerobic environments results in accumulation of 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, isomers with other combinations of partially or fully reduced nitro groups, and azoxy dimers (11, 21, 32, 33). Cometabolic oxidation of TNT to 2,4,6-trinitrobenzyl alcohol and 2,4,6-trinitrobenzoate has also been reported for propane-grown Mycobacterium vaccae (30), but putative enzymatic steps or intermediates in these reactions have not been described.
Unlike TNT, dinitrotoluenes are known to be growth substrates for
aerobic bacteria (24). In the initial steps of the metabolic pathways, nitrite is apparently released from intermediates of the
hydroxylation or dioxygenation reactions by 
,
elimination. Thus,
for aerobic metabolism of TNT, release of nitrite from the aromatic
ring appears to be a critical requirement for further metabolism.
Mineralization of TNT by TOL transformants of Pseudomonas sp. clone A has been reported (6, 11), suggesting that
cometabolic hydroxylation reactions may indeed facilitate nitrite
release. 2,4-Dinitrotoluene has been suggested as a TNT
by-product, but mass balance confirmation of nitrite
release was not provided (11). Likewise, a
Mycobacterium strain reduced TNT to H-TNT and apparently
released nitrite (31), but subsequent studies revealed
false-positive reactions of the Griess reagent used for colorimetric nitrite quantitation (32), making the
assessment of the nitrite release mechanism equivocal.
Pentaerythritol tetranitrate (PETN) reductase purified from
Enterobacter cloacae has also been reported to catalyze
TNT transformation by aromatic ring reduction and to release nitrite,
again on the basis of the use of the Griess reagent (8, 9).
In these previous studies, the carbon-containing metabolites associated
with nitrite release were not identified, leaving the mechanism of
nitrite release unresolved.
Recently, we have worked with the flavoprotein xenobiotic reductases XenA from Pseudomonas putida II-B and XenB from P. fluorescens I-C (1, 2). Along with PETN reductase and other enzymes, XenA and XenB are members of an evolutionarily related family of NAD(P)H-dependent flavoprotein oxidoreductases. Although XenA and XenB have high primary sequence identity, suggesting similar three-dimensional structures, they exhibit different selectivities for removal of the nitro groups from nitroglycerin. These differences in catalytic selectivity extend to reactions with TNT. While XenA catalyzes only nitro group reductions, XenB transforms TNT via nitro group reduction and direct reduction of the aromatic ring. Using ion pair high-pressure liquid chromatography (HPLC), ion chromatography, liquid scintillation counting, optical, and mass spectral techniques, we have detected 13 TNT products resulting from XenB-initiated TNT transformations. Among the reactions, a nonenzymatic reaction of the hydroxylamino-dinitrotoluene (HADNT) isomers and 2H-TNT · H+ results in nitrite release and the formation of various isomers with proposed chemical structures corresponding to that of amino-dimethyl-tetranitrobiphenyl. Since the same products are observed after transformation of TNT by P. fluorescens I-C, potential accumulation of these novel biphenyl dimers in the environment is indicated.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. Nitroglycerin was provided by Olin Corporation (Baraboo, Wis.). TNT was obtained from Chem Service (West Chester, Pa.). 2,4-Dinitrotoluene, 2,6-dinitrotoluene, 1,3-dinitrobenzene, nitrobenzene, and the three nitrotoluene isomers were purchased from Aldrich Chemical Company (Milwaukee, Wis.). [U-14C]TNT (21.58 mCi/mmol; purity, greater than 95%, as determined by thin-layer chromatography) was purchased from ChemSyn Science Labs (Lenexa, Kans.). Dansyl chloride was purchased from Molecular Probes (Eugene, Oreg.). Isotopically labeled 18OH2 (97% 18O content) was obtained from ICON (Summit, N.J.). All other chemicals were analytical grade or better.
Chemical syntheses. The HADNT isomers were chemically synthesized by using zinc dust and ammonium chloride (19) and were purified by HPLC as described below. H-TNT was chemically synthesized by using sodium borohydride (11, 16) and was purified by HPLC as described below. Isomers of 2H-TNT · H+ were also produced by reduction of TNT with sodium borohydride and were purified by HPLC.
Dansyl chloride was prepared as a 100 mM solution in acetonitrile. Dansylation reactions were performed by mixing 15 µl of dansyl solution, 100 µl of NaHCO3 (50 mM, pH 8.3), and 50 µl of HPLC-purified compounds resuspended in water. Reactions were carried out in the dark at room temperature. After 30 min of incubation, the dansylation reaction was stopped by addition of 25 µl of 0.1 N NaOH (10). For thin-layer chromatography, HPLC-purified compounds were resuspended in acetonitrile:water (1:1, vol/vol) and spotted onto silica gel thin-layer chromatography plates. Each silica plate was sprayed with NaNO2 (0.1% [wt/vol] solution in 1 N HCl), followed by N-1-naphthylethylenediamine HCl (0.04% [wt/vol] solution prepared in methanol). The plate was air dried and then heated at 100°C for 1 h and exposed to UV light (27).Bacterial strains, culturing methods, and whole-cell assays. The bacterium used in this study, P. fluorescens I-C, was isolated (2) from nitroglycerin-contaminated soil obtained from the Badger Army Ammunition Plant (Baraboo, Wis.) on the basis of its ability to tolerate high concentrations of nitroglycerin in the growth medium. For whole-cell assays, P. fluorescens I-C was grown aerobically in Luria-Bertani medium at 30°C. After overnight incubation, the cells from 1 ml of the medium were isolated by centrifugation at 14,000 × g, washed twice with 100 mM phosphate buffer (pH 7), and resuspended in 1 ml of the same buffer. A 500-µl aliquot of 0.56 mM TNT dissolved in deionized water was added to the culture, and TNT transformation products were determined by HPLC.
Enzyme reactions. XenB was purified by standard chromatographic methods and was characterized as previously reported (2). One unit of XenB activity was defined as the amount of enzyme that catalyzed consumption of 1 µmol of NADPH per min. Reactions were carried out in 1 ml of 100 mM phosphate (pH 7.0) in the presence of 300 µM nitro-containing substrate and 130 µM NADPH. TNT transformation reactions were performed in 1.5-ml polyethylene microcentrifuge tubes containing 900 µl of 100 mM phosphate (pH 7.0), 100 nmol of TNT, and 300 nmol of NADPH. The reactions were initiated by the addition of purified XenB to give 5.4 µg of enzyme per reaction mixture.
HPLC methods. TNT metabolites were separated by ion pair chromatography by using a Beckman (Fullerton, Calif.) HPLC equipped with a Nova-Pak radial compression C18 column (bead size, 4 µm; 5 by 100 mm; Waters Chromatography, Milford, Mass.). The ion pair reagent tetrabutylammonium dihydrogen phosphate (pH 7.5) was added to both deionized water (aqueous buffer) and acetonitrile (acetonitrile buffer) to a final concentration of 5 mM. The initial composition of the mobile phase was 75% aqueous buffer and 25% acetonitrile buffer. This composition was maintained for 1 min. The percentage of acetonitrile buffer was then linearly increased to 32% over 12.2 min, linearly increased to 64% over 10.5 min, and increased stepwise to 90%. The 90% acetonitrile buffer composition was maintained for 2.3 min. At the end of the separation protocol, the column was reequilibrated to 25% acetonitrile buffer over 5 min. TNT and metabolites were detected by UV-visible absorption spectrometry by using a diode array detector. Metabolites derived from aromatic ring reduction were detected at 477 nm, while metabolites derived from nitro group reduction were detected at 230 nm. The retention times observed for TNT and other metabolites by the ion pair HPLC method and elution protocol described above are summarized in Table 1.
TNT metabolites resulting from dimerization reactions (Table 1, compounds 11 and 12) were not completely separated by the ion pair HPLC method and were analyzed by performing a different gradient elution with 20 mM ammonium acetate buffer (pH 7.5) and acetonitrile. During separation, an initial mobile phase composition of 100% ammonium acetate buffer was maintained for 1 min. The percentage of acetonitrile was linearly increased to 20% over 2 min and maintained for 5 min, linearly increased to 25% over 2 min and maintained for 2 min, increased to 35% over 0.5 min and maintained for 7.5 min, and increased to 90% over 0.5 min and maintained for 14.5 min.Ion chromatography. Nitrite was detected and quantified by using an LC20 ion chromatograph (Dionex, Sunnyvale, Calif.) equipped with a GP40 gradient pump, an ED40 electrochemical detector, an IonPac AG11 guard column (4 by 50 mm), an IonPac ATC-1 anion trap column, an ASRS-Ultra 4-mm suppression system, and an IonPac AS11 anion-exchange column (4 by 250 mm). NaNO2 was used as a standard for calibration curves.
ESI-MS analysis. Electrospray ionization mass spectral (ESI-MS) data were obtained with a Perkin-Elmer Sciex API 365 triple quadrupole spectrometer at the Mass Spectrometry Facility of the University of Wisconsin Biotechnology Center. When the TNT metabolites were recovered after ion pairing separation, the fractions collected were dried with a speed vacuum, resuspended in acetonitrile, and then separated from the pairing reagent by HPLC by the ammonium acetate method described above. Metabolites recovered after HPLC separation by the ammonium acetate method were dried and resuspended on acetonitrile-water (1:1, vol/vol) for injection into the spectrometer.
Liquid scintillation counting.
[U-14C]TNT was
mixed with TNT in acetonitrile to produce a 10 mM solution with a
specific activity of 8,770 dpm/nmol. Ten-microliter portions of this
solution were added to enzyme reaction mixtures, giving a total of 100 nmol of TNT per reaction mixture. The distribution of 14C
in TNT and the various metabolites was evaluated by injecting 100 µl
of a reaction mixture into the HPLC column and collecting 0.5-ml
aliquots of the HPLC eluent throughout the entire HPLC run. A
scintillation cocktail (4.5 ml; ScintiSafe Econo 2; Fisher) was added
to the vials, and the counts per minute were determined with a Packard
Tri-Carb 2100TR liquid scintillation analyzer. Individual scintillation
vials were counted either for 1 min or to achieve an E2
value of more than 2%. The 14C elution profile was
correlated with the retention times of TNT and the known metabolites to
determine the relative percentage of each compound in the enzyme
reaction mixtures. In addition, the 14C elution profile
was used to define the retention times and quantities of unknown metabolites.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reaction of purified XenB with TNT.
In this work, we focused
on characterization of metabolites produced by the reaction of purified
XenB with TNT under either aerobic or anaerobic conditions. In
addition, chemically synthesized compounds corresponding to the
metabolites isolated from the enzyme reaction mixtures were used to
investigate the potential contributions of successive enzymatic
reductions and abiotic reactions. A summary of the TNT metabolites
detected is presented in Table 1, while Fig.
1 shows the products of XenB-catalyzed
reduction of TNT. Figure 1 also shows a proposed mechanism for abiotic
reactions leading to the release of nitrite and the formation of
dimeric compounds that are proposed here to be various isomers of
amino-dimethyl-tetranitrobiphenyl.
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Release of nitrite from TNT.
Ion chromatography was used to
demonstrate unambiguously and to quantify the release of nitrite from
TNT during XenB-catalyzed reactions (Fig.
2). From the 100 nmol of TNT originally
present in the aerobic enzyme reaction mixtures, ~28 nmol of nitrite
was recovered after 5 h and ~50 nmol was recovered after 24 h. With anaerobic enzyme reaction mixtures, ~18 nmol of nitrite was
recovered after 5 h and ~48 nmol was recovered after 24 h.
Nitrite was also observed during aerobic incubation of TNT with
P. fluorescens I-C cells (Fig. 2), with ~23 nmol of
nitrite recovered after 5 h and ~37 nmol recovered after 24 h. In all the experiments, at least 50% of the TNT had been
transformed after only 10 min of incubation and about 90% had been
transformed after 30 min. Thus, a primary goal of this work was to
characterize the TNT metabolites that were produced by the nitrite
release reaction.
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, aerobic transformation by XenB;
, anaerobic transformation by XenB; 
, aerobic transformation by
P. fluorescens I-C.
Products observed from aromatic ring reduction.
Upon addition
of TNT, reaction mixtures containing XenB changed from colorless to red
and then to yellow. According to previous descriptions (31,
32), these changes suggested successive formation of H-TNT,
2H-TNT, and various 2H-TNT · H+. HPLC analyses of
the XenB reaction mixtures demonstrated transient accumulation of
compound 2, which had an HPLC retention time of 23.6 min (Table 1) and
an optical spectrum (
max, ~480 nm [31, 32]) indistinguishable from that of chemically synthesized
H-TNT. After resuspension of compound 2, negative-mode ESI-MS revealed an m/z value of 228, which corresponded to the chemical
formula of the H-TNT anion (Table 1). Control experiments performed
with TNT, NADPH, and no enzyme showed no conversion of TNT,
demonstrating that XenB was required to catalyze the formation of
compound 2. Thus, compound 2 was identified as H-TNT, a transient
product of the NADPH-dependent reaction of XenB and TNT.
max of 236 nm,
and a visible max of 449 nm; compound 4, with a
retention time of 10.1 min, a UV max of 262 nm, and a
visible max of 492 nm; compound 5, with a retention time
of 13.4 min, a UV max of 262 nm, and a visible
max of 466 nm; and compound 6, with a retention time of
17.7 min, a UV max of 262 nm, and a visible
max of 487 nm) (Table 1; Fig. 1). These products had
UV-visible spectra similar to those reported for metabolites arising
from reduction of H-TNT (8, 32) and subsequently identified
(32) as various tautomers of 2H-TNT · H+.
Negative-mode ESI-MS analyses of the HPLC-purified metabolites revealed
an m/z value of 230 for each of the four compounds, which corresponded to the molecular mass of 2H-TNT · H+
(Table 1).
Enzyme reactions in which chemically synthesized compound 2 was used as
the substrate resulted in rapid accumulation of compounds 3 through 6. In contrast, control reaction mixtures that contained compound 2, NADPH, and no enzyme yielded the previously demonstrated (11,
32) reverse transformation of H-TNT to TNT (~98% conversion after 12 h) and no accumulation of compounds 3 through 6. These results demonstrate that XenB also catalyzes reduction of H-TNT to
2H-TNT.
Further studies of compounds 3 through 6 revealed that their
interconversion was not enzymatic and that compound 5 was the most
stable of these four metabolites. Thus, when HPLC-purified compound 3 (i.e., compound 3 chemically synthesized by reduction of TNT with
sodium borohydride, collected from an HPLC eluent, and dried with a
speed vacuum) was resuspended in phosphate buffer (100 mM, pH 7.0) and
reinjected into the HPLC after 3 h, compound 3 (6% ± 1%
[average ± standard deviation based on at least two experiments]), compound 5 (79% ± 3%), and compound 6 (15% ± 2%) were detected. Similarly, resuspension and reinjection of compound 4 into the HPLC resulted in the appearance of compounds 3 (6% ± 2%), 4 (20% ± 1%), 5 (62% ± 2%), and 6 (12% ± 3%). Resuspension and
reinjection of compound 5 resulted in mainly compound 5 (81% ± 4%)
and smaller amounts of compounds 3 (5% ± 1%) and 6 (14% ± 2%).
Finally, reinjection of compound 6 revealed mainly compound 5 (81% ± 2%) and smaller fractions compounds 3 (5% ± 1%) and 6 (14% ± 1%). When HPLC-purified compounds 3 through 6 were
individually used as substrates for XenB reactions, no further
metabolites were detected and the distribution of HPLC peak areas among
the resultant products (compounds 3 through 6) was nearly
indistinguishable from that obtained in the absence of XenB.
Taken together, these experiments demonstrated that XenB can catalyze
the reduction of TNT (compound 1) to H-TNT (compound 2) and the
reduction of H-TNT to 2H-TNT. Further rearrangements among the various
2H-TNT tautomers (compounds 3 through 6) was nonenzymatic. These
results are summarized in Fig. 1. Our data were not sufficient to
determine the exact chemical structures of compounds 3 through 6. Nevertheless, the results show that compounds 3 through 6 are related
to each other by reversible reactions and that their relative
concentrations in the reaction mixtures reach chemical equilibrium in
either the presence or the absence of XenB and NADPH.
Products observed from nitro group reduction. In addition to the products derived from aromatic ring reduction, the XenB-catalyzed reaction with TNT yielded substantial amounts of 2HADNT (compound 7, with a retention time of 11.6 min) and 4HADNT (compound 8, with a retention time of 12.1 min) (Table 1; Fig. 1), and compound 8 was the major product. Identification of these metabolites was based on the following experimental evidence: (i) the HPLC retention times of the enzyme products closely matched those of chemically synthesized compounds 7 and 8; (ii) the UV-visible spectra of the enzyme products matched those of the chemically synthesized compounds; and (iii) negative-mode ESI-MS revealed an m/z value of 212 for both compound 7 and compound 8, which corresponded to the predicted mass of the anion of the two possible HADNT isomers (Table 1). The individual isomers were assigned to the corresponding HPLC peaks on the basis of the higher yield of compound 8 obtained from the chemical synthesis (19) and on the basis of differences in the UV-visible spectra reported for the two isomers (15).
For longer incubation periods, small amounts of 2-amino-4,6-dinitrotoluene (compound 9, with a retention time of 15.0 min) and 4-amino-2,6-dinitrotoluene (compound 10, with a retention time of 15.5 min) were also identified on the basis of the similarity of the HPLC retention times with those of available standards and on the basis of the m/z value of 196 determined by ESI-MS analyses (Table 1).Use of [U-14C]TNT.
[U-14C]TNT was
used to ascertain the mass balance for the metabolites produced by
XenB. Figure 3 shows the time course for
distribution of 14C during a typical aerobic enzyme
reaction. For simplicity, the 14C counts were treated as
counts from five groups of metabolites, reflecting unreacted TNT
(compound 1), metabolites from aromatic ring reduction (compounds
2 through 6), metabolites from nitro group reduction (compounds 7 through 10), dimerization products (compounds 11 and 12), and two
unidentified metabolites having retention times of 1.5 to 4.0 min
(compounds 13 and 14). About 6 to 10% of 14C was
associated with compounds 13 and 14, and this proportion did not change
substantially during the course of the aerobic reactions. Recovery of
14C during HPLC separations was greater than 80%
throughout the entire reaction period.
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metabolites from aromatic ring reduction (compounds 2 through 6); 
,
dimerized products (compounds 11 and 12); ESI-MS of compounds 11 and 12 produced by XenB. For better separation of metabolites with retention times of 24.5 to 25.5 min (Table 1), a different HPLC method with ammonium acetate and acetonitrile was used, as described in Materials and Methods. With this method, three TNT metabolites with an m/z of 376 (Table 1, compound 12) eluted with retention times of 23.5, 23.8, and 24.1 min. The metabolite exhibiting the largest absorption peak eluted at 23.8 min. In addition, a metabolite with an m/z of 405 (compound 11) eluted at 24.3 min. The molecular mass of compound 11 corresponded to that of previously well-characterized azoxydinitrotoluene dimers (11, 21, 33). In contrast, the molecular mass of compound 12 suggested an unknown compound whose mass was consistent with dimerization of TNT metabolites in which nitrite was also released (Table 1; Fig. 1).
An m/z of 376 is consistent with the molecular anion of a compound with an odd number of nitrogen atoms (18). In principle, the mass of compound 12 could be accounted for by an azoxy dimer having one nitrite group replaced by a hydroxyl group. However, when compound 12 was formed in buffer prepared by using 18O-enriched water, an m/z of 376 was still obtained, showing that replacement of a nitrite group by a hydroxyl group was not required to form compound 12. Furthermore, the 18O experiment suggested that compound 12 did not contain a nitroso functional group whose O atom would likely exchange with water.Combined chemical and enzymatic synthesis of compounds 11 and 12. A mixture of compounds 7 and 8 was chemically synthesized and used as the substrate for an aerobic XenB reaction. After 24 h, compounds 7 and 8 were completely consumed, and only compound 11 was observed by combined HPLC and ESI-MS analyses. A control experiment conducted with compounds 7 and 8 and without XenB also gave only compound 11, showing that formation of compound 11 was not enzyme catalyzed. Since azoxy dimers are formed from HADNT isomers (33), compound 11 was assigned to represent all isomeric azoxy dimers having an m/z of 405 that were observed in this work (Table 1). When similar aerobic XenB reactions were undertaken by using the HPLC-purified, individual, aromatic ring reduction products (compounds 3 through 6) as the substrates, no further enzymatic transformation was observed (see above), and notably, neither compound 11 nor 12 was produced.
The results in Fig. 3 suggested that compound 12 may have arisen from combination of the products of aromatic ring reduction and nitro group reduction. Thus, by combination of chemically synthesized and HPLC-purified compounds 7 and 8 with enzymatically synthesized and HPLC-purified compound 5 (the major species of compounds 3 through 6), compounds 11 and 12 were produced, and compound 12 was the major product. In aerobic reactions in which the synthesized precursors were used, the rates of formation of compound 12 were similar regardless of whether XenB or NADPH was present. Moreover, in experiments performed with an excess of chemically synthesized compounds 7 and 8, the amount of compound 12 formed was proportional to the amount of compound 5 present (Fig. 4). Direct quantification of the nitrite released during the reaction of compound 5, with compounds 7 and 8 was unfortunately not possible because at high concentrations of compounds 7 and 8, a peak resulting from these compounds was not fully resolved from the nitrite peak. Nevertheless, the results of these experiments show that compound 12 was formed by the nonenzymatic reaction of an HADNT (compound 7 or 8) and a metabolite derived from aromatic ring reduction (compound 3, 4, 5, or 6), and the molecular weight of compound 12 is consistent with the release of a nitro group during the reaction. Since both compounds 7 and 8 were consumed in the reaction, at least two isomers of compound 12 are likely. Furthermore, the observed solution equilibrium involving compounds 3 through 6 did not permit identification of which compound reacted to give compound 12, but if more than one compound did react, additional isomers of compound 12 would be likely.
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, 76 nmol of compound 5 added.
Anaerobic transformation of TNT by XenB.
Figure
5 shows the time course for distribution
of 14C during a typical anaerobic enzyme reaction. As in
the aerobic mass balance study (Fig. 3), the 14C counts
were treated as counts for five groups of metabolites. In this
experiment, the percentage of total 14C recovered from HPLC
separation was greater than 99% during the reaction period. After 80 min of incubation, 62% of the 14C was in products derived
from aromatic ring reduction (mostly compounds 3, 5, and 6), 25% was
in products derived from nitro group reduction (compounds 7 and 8, 21%; compounds 9 and 10, 4%), and ~13% was in two unidentified
compounds (compounds 13 and 14; retention times, 1.5 to 4.0 min).
Notably, compounds 11 and 12 were not produced under anaerobic
conditions and accounted for only 2% of the 14C after 80 min.
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TNT transformation by whole cells. When P. fluorescens I-C cells grown on Luria-Bertani medium and resuspended in 100 mM phosphate buffer (pH 7) were amended with TNT, products derived from both aromatic ring and nitro group reduction were detected. As observed with the purified enzyme, the culture medium immediately changed from colorless to red and then to yellow upon the addition of TNT. Figure 1 shows that nitrite was released in the whole-cell reactions, albeit in lower amounts than those observed in the purified enzyme reactions. After 45 min of incubation, nitrite accumulations in whole-cell and enzyme reaction mixtures were 2.5 and 18 nmol, respectively, while after 24 h of incubation, nitrite accumulations in whole-cell and enzyme reaction mixtures were 37 and 50 nmol, respectively. HPLC separation with detection at 477 nm revealed transient formation of compound 2 and accumulation of compounds 3 through 6, while detection at 230 nm revealed accumulation of compounds 7 and 8 and their subsequent conversion to compounds 9 and 10. Accumulation of the latter two metabolites was substantially greater in the whole-cell reaction mixtures than in the enzyme reaction mixtures. Furthermore, both compound 11 and compound 12 were detected in the whole-cell experiments, although accumulation of these compounds was less than that observed in the enzymatic reactions. In whole-cell reaction mixtures, accumulation of compound 12 after 24 h of incubation was 33% less than that in enzymatic reaction mixtures, a difference that correlates well with the observed difference in nitrite accumulation (e.g., there was 26% less nitrite in whole-cell reaction mixtures). The decrease in the accumulation of nitrite and compound 12 was likely due to more extensive conversion of compounds 7 and 8 to compounds 9 and 10, resulting in decreased availability of intermediates required for synthesis of compounds 11 and 12.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our results show that XenB has the ability to catalyze transformation of TNT by aromatic ring reduction and by nitro group reduction. Furthermore, our results show that nonenzymatic reaction of the products leads to release of nitrite under oxidizing conditions (in the presence of either O2 or NADP+) and to accumulation of dimers thought to be isomers of amino-dimethyl-tetranitrobiphenyl. Since P. fluorescens I-C containing XenB produced the same products as those observed with the purified enzyme, it is possible that amino-dimethyl-tetranitrobiphenyls may be produced in natural environments. This suggested pathway provides a new focus for enzymatic enhancement of TNT bioremediation.
Nitrite release from TNT initiated by XenB-catalyzed reduction. Like XenB, PETN reductase from E. cloacae PB2 has been reported to transform TNT by both aromatic ring reduction and nitro group reduction (8). As determined by colorimetric assays, nitrite release was reported in PETN reductase studies and in other studies (6-8, 31). However, no major carbon-containing products of the denitration reaction were identified in these previous studies, which ultimately limited both the insight into the mechanism of nitrite release and the prospects for improving the putative biotransformations. By using purified XenB, ion chromatography, 14C tracer methods, and reactions of chemically synthesized precursors, we found that up to 0.50 nmol of nitrite can be released per nmol of TNT consumed (Fig. 1). On the basis of enzymatic and chemical characterizations, it now appears that compounds 3 through 6 and compound 7 or 8 are required for the nitrite release reaction described here. The half-integral stoichiometry for the amount of nitrite released relative to the amount of TNT consumed supports the proposed nonenzymatic dimerization mechanism for nitrite release (Fig. 1). On the basis of these results, other transformations would be required to increase the stoichiometry of nitrite release during TNT metabolism.
Proposed mechanism for nitrite release and dimerization. Our results show that XenB catalyzes reduction of TNT to compounds 3 through 6 and compounds 7 through 10. In contrast, the predominant end product isolated from the reaction mixtures has been proposed to be compound 12, an amino-dimethyl-tetranitrobiphenyl. A mechanism accounting for the formation of compound 12 is shown in Fig. 1. In this proposal, dimerization is initiated by the interaction of resonance-stabilized aryl nitrenium cation and aryl carbanion species. Thus, ion pairing and hydrophobic stacking interactions may be implicit features of this reaction. Acid-catalyzed decomposition of an aromatic hydroxylamine, which is the first step of the Bamberger rearrangement (17), provides a precedent for the proposed formation of a resonance-stabilized aryl nitrenium cation and is consistent with the requirement for an HADNT isomer during the formation of compound 12. Furthermore, compound 2 and compounds 3 through 6 are resonance-stabilized carbanionic intermediates due to the presence of the multiple nitro group substituents, which permits extensive delocalization of electron density from the added hydride equivalents. In this work, the dihydride level intermediates compounds 3 through 6 were shown to also participate in the formation of compound 12.
As noted above, compound 12 likely represents a variety of possible isomers. Figure 1 shows a representative dimerization reaction that gives the sterically least-hindered product; analogous reactions can be drawn to potentially give five other isomers. Formation of the C---C bond between C---4 of 2H-TNT · H+ and C---3 of the aryl nitrenium cation would place a nitro group originally from 2H-TNT · H+ in a position suitable to be eliminated as nitrite, while rearrangement of the imine group would lead to rearomatization of the aromatic ring originally from HADNT. Elimination of nitrite as shown in Fig. 1 would be favorable, as nitrite is an excellent leaving group at neutral pH. Indeed,, elimination of
nitrite has been observed in a number of other relevant enzyme
reactions, notably nitrobenzene dioxygenase (20),
1,3-dinitrobenzene dioxygenase (5), 2-nitrotoluene
dioxygenase (12), 2,4-dinitrotoluene dioxygenase (23,
26, 28), and nitrophenol oxygenase (25) reactions.
Furthermore, rearrangement of the imine with subsequent rearomatization
should be a highly favorable reaction leading to the formation of an
aryl amine functional group. Due to a reduced pKa value
caused by the ring nitro substituents, the aryl amine should readily
react with an acylating agent, such as dansyl chloride, at neutral pH
or with NaNO2 and N-1-naphthylethylenediamine
HCl under more extreme conditions. Thus, the quantitative conversion of
amino-dinitrotoluenes and compound 12 and the lack of reactivity of TNT
with these diagnostic reagents provide chemical evidence for the
presence of an aryl amine group.
The final rearomatization reaction and the formation of compound 12 would require 2e oxidation and H+ release.
This reaction would be analogous to the well-documented conversion of
H-TNT to TNT (31), a process that requires 2e
oxidation and H+ release. As shown in Fig. 6B, this
reaction can be mediated by either O2 or NADP+.
In principle, a variety of other oxidants could also function in this
final oxidation step. The difference between the rate of nitrite
release and the rate of formation of compound 12 under either aerobic
or anaerobic conditions could in part be due to a faster
rearomatization reaction in the presence of O2 than in the
presence of other oxidants or due to the effects of the different concentrations of the oxidants on the bimolecular reaction.
Enzymology of TNT metabolism. XenB is a member of a family of NAD(P)H-dependent flavoprotein xenobiotic reductases (1). Primary sequence analysis of two members of this family, XenA and XenB, revealed 34% primary sequence identity and 51% similarity, suggesting that these enzymes may have related three-dimensional structures. However, catalytic characterizations revealed substantial differences in the regioselectivity for nitrite removal from nitroglycerin and the reduction of cyclohexenone (1, 2). In this work, initial characterizations revealed that XenA had ~20% of the reactivity of XenB with TNT when preparations were indirectly assayed by determining NADPH consumption and that XenA gave only products derived from nitro group reduction, while XenB could transform TNT into compounds 2 through 6 and compounds 7 through 10.
The ability of other xenobiotic reductases to release nitrite via the dimerization reaction described here may depend on the ability to match the products given by XenB, which include products of both aromatic ring reduction and nitro group reduction. Alternatively, xenobiotic reductases capable of producing primarily hydroxylamino metabolites may primarily yield azoxy dimers in aerobic environments, while xenobiotic reductases capable of producing primarily aromatic ring reduction metabolites offer alternative, although currently poorly understood, possibilities for TNT bioremediation. Studies with P. fluorescens I-C revealed increased accumulation of amino-dinitrotoluenes compared to that observed in the purified enzyme reactions, suggesting the presence of another enzyme that enhances production of these metabolites. The formation of amino-dinitrotoluenes would also contribute to the observed decreases in the amount of compound 12 and nitrite release, since a required substrate would be consumed by an alternative reaction.Environmental implications. The denitration mechanism proposed here provides insight into a potential constraint on microbial utilization of TNT as a nitrogen source, as nitrite release would depend on the availability of both compounds 3 through 6 and compound 7 or 8. Rapid conversion of TNT to amino-dinitrotoluenes, as observed in the whole-cell experiments, would also effectively minimize the nitrite release reaction, which could explain why Rhodococcus erythropolis HL PM-1 failed to utilize TNT as a nitrogen source even though the two required TNT reduction pathways were present (32). Furthermore, production of the precursors compounds 3 through 6 and compound 7 or 8 would require at a minimum consumption of seven equivalents of NAD(P)H in order to produce one equivalent of utilizable nitrogen (e.g., four equivalents consumed by XenB to yield compound 12 and three equivalents consumed by nitrite reductase [EC 1.6.6.4] to convert nitrite to ammonia). This substantial energy demand and the limited nitrite release may effectively prevent utilization of TNT as a sole source of nitrogen by the mechanism proposed in Fig. 1. However, regardless of whether TNT can be used as a source of nitrogen, the results also suggest that polynitrated biphenyl compounds may be formed in the environment from TNT as a consequence of reductive cometabolism. While these compounds have not been reported in previous nitroaromatic transformation studies, the similar chemical reactivities of nitrite and chlorine suggest that various isomers of compound 12 may have properties similar to those of polychlorinated biphenyls, including insolubility, toxicity (4, 14), and recalcitrance to further bioremediation (3, 22, 29). Whether these compounds can be further transformed in either the laboratory or the natural environment remains to be determined.
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ACKNOWLEDGMENTS |
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This work was partially supported by grant MCB 9733734 from the National Science Foundation (to B.G.F.) and contract DAA21-93-C-1034 from the U.S. Army ARDEC (to G.H.C.). Additional support was provided by the College ot Agricultural and Life Sciences (to G.H.C.) and the College of Engineering (to D.R.N.), University of Wisconsin-Madison.
We thank A. Harms (University of Wisconsin Biotechnology Center) for expert help with the ESI-MS analysis.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, 1415 Engineering Drive, Madison, WI 53706. Phone: (608) 263-7783. Fax: (608) 262-5199. E-mail: noguera{at}engr.wisc.edu.
Present address: Dupont Experimental Station, Wilmington, DE
19880-0328.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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