Applied and Environmental Microbiology, May 2004, p . 2854-2860, Vol . 70, No . 5

Nitrite Elimination and Hydrolytic Ring Cleavage in 2,4,6-Trinitrophenol (Picric Acid) Degradation

Klaus W . Hofmann, Hans-Joachim Knackmuss, and Gesche Heiss^*

Institute of Microbiology, University of Stuttgart, 70569 Stuttgart, Germany

Received 3 June 2003/ Accepted 10 February 2004

It was previously established that two hydrogenations take place in the initial attack on TNP (6, 7, 8, 11) (Fig . 1A, panel 1) . In Rhodococcus opacus HL PM-1, TNP is hydrogenated at the aromatic nucleus by the hydride transferase II (HTII) encoded by npdI (Fig . 1B) and the NADPH-dependent F₄₂₀ reductase (NDFR) encoded by npdG . The hydride Meisenheimer complex of TNP (H^–-TNP) (Fig . 1A, panel 2) thereby formed is further hydrogenated by the hydride transferase I (HTI) encoded by npdC and the NDFR, producing the dihydride Meisenheimer complex of TNP (2H^–-TNP) (panel 3a) . In Nocardioides simplex FJ2-1A, the same reactions take place except that a single hydride transferase performs both hydrogenations (7) .

 
More than a decade ago, 4,6-dinitrohexanoate was identified as a dead-end metabolite of TNP degradation resulting from two hydride transfers to TNP . This observation coincided with the hypothesis that 2H^–-TNP was a dead-end metabolite of TNP degradation (11) . Much later it was suggested that nitrite is eliminated from 2H^–-TNP to produce the hydride Meisenheimer complex of DNP (H^–-DNP) in N . simplex FJ2-1A (7) . Hence, 2H^–-TNP is a metabolite of productive TNP degradation . Detection of H^–-DNP suggests that the pathways for TNP and DNP degradation converge, although indisputable identification of H^–-DNP is still required to confirm the hypothesis . NpdH, a gene product of the npd gene cluster of R . opacus HL PM-1, was recently shown to convert 2H^–-TNP to an unknown product X (Fig . 1A, panel 3b), which was preliminarily suggested to be a tautomer of protonated 2H^–-TNP (8); this evoked the issue of which form of 2H^–-TNP is the substrate for nitrite release .

To address these issues, three enzymes in the TNP degradation pathway were identified and the metabolites were confirmed . Hence, evidence is supplied for the convergent TNP and DNP (Fig . 1A, panel 4) degradation pathways, with H^–-DNP (panel 5) as the first common metabolite . We have shown that four unusual catabolic reactions (three ring hydrogenations and a hydrolytic ring fission) take place in the upper TNP degradation pathway .

Escherichia coli BL21(DE3) (pNTG11) expressing npdH, E . coli JM109 (pNTG6) expressing npdC, and E . coli TOP10 (pDMW10) expressing npdG (8) were grown at 37°C in Luria-Bertani medium containing 100 µg of ampicillin ml^–1 . Overnight cultures were inoculated into Luria-Bertani medium and grown at 37°C to an optical density at 600 nm of 0.4 . Cultures were induced for 4 h at 30°C with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) .

Preparation of cell extracts.
About 20 g of wet cells of N . simplex FJ2-1A or 2 g of wet cells of the E . coli strains was suspended in 50 mM Tris-HCl (pH 7.5) and lysed with a French press as described before (8) . The protein concentration was determined with a dye reagent concentrate (protein assay; Bio-Rad, Munich, Germany) by the method of Bradford (5) .

Enzyme purification.
npdH, npdC, and npdG from R . opacus HL PM-1 were expressed in E . coli BL21(DE3) (pNTG11), E . coli JM109 (pNTG6), and E . coli TOP10 (pDMW10), respectively, and the proteins were purified as His-tag fusion proteins by Ni-nitrilotriacetic acid affinity chromatography as described before (8) . Imidazole was removed from the enzyme-containing fractions with pD10 desalting columns (Amersham Pharmacia, Freiburg, Germany) . Samples were concentrated by ultrafiltration (Vivaspin 2; Vivascience AG, Hanover, Germany) .

The nitrite-eliminating enzyme was purified from N . simplex FJ2-1A at 4°C by fast-performance liquid chromatography (LC) (LCC 500 controller, 500 pump, UV-1 monitor, REC-482 recorder, and FRAC autosampler; Pharmacia, Uppsala, Sweden) . The cell extract (340 mg of protein) was passed through a Q Sepharose column (HP HR 16/10; Pharmacia) preequilibrated with basic buffer (50 mM Tris-HCl [pH 7.5]) at a flow rate of 1 ml min^–1 . The activity was eluted from the column with a linear gradient of 0 to 1 M NaCl (200 ml) in basic buffer at 0.25 M NaCl . Ammonium sulfate (1 M) was added to the active fractions and applied to a Phenyl Superose column (HR10/10; Pharmacia) preequilibrated with the same buffer . Enzyme was eluted with a linear gradient (65 ml) of 1 to 0 M ammonium sulfate in basic buffer at 0.41 M (NH₄)₂SO₄ and a flow rate of 0.5 ml min^–1 . Active fractions were applied to a gel filtration column (Superdex 200 Prep-Grade; Pharmacia) (1 by 30 cm), and the enzyme was eluted with basic buffer at a flow rate of 1 ml min^–1 . The hydrolase was purified from N . simplex FJ2-1A as described above except that elution from the Q Sepharose column was at 0.33 M NaCl and from the Phenyl Superose HR column was at 0.54 M (NH₄)₂SO₄ .

The molecular mass of native proteins was determined using a gel filtration calibration kit (Amersham Pharmacia) . Purity and molecular mass of protein subunits were determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) with a G1025A Hewlett-Packard LD-TOF system (GSG Mess- und Analysegeräte Vertriebsgesellschaft mbH, Karlsruhe, Germany) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% (vol/vol) polyacrylamide gel by the method of Laemmli (9) on a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad) .

Enzyme assays.
Enzyme assays were performed with a Cary 50 biospectrophotometer controlled by Cary WinUV Biopackage software (Varian, Mulgrave, Australia) . Reactions with the tautomerase were performed as previously described for NpdH (8) .

The activity of the nitrite-eliminating enzyme was assayed by measuring the increase in absorbance of H^–-DNP at 450 nm or repeated recording of UV-visible spectra between 280 and 600 nm in 1-min cycles . The test was conducted with 50 mM Tris-HCl (pH 8.0) containing 0.1 mM 2H^–-TNP and 6 µg of nitrite-eliminating enzyme . Specific activities were calculated by using the experimentally determined extinction coefficient of 944 M^–1 cm^–1 (H^–-DNP at 450 nm; 20°C; pH 8.0) .

The activity of HTI with respect to H^–-DNP was measured as described before (8) except that 100 µM H^–-DNP was the substrate instead of H^–-TNP . The decrease in absorbance of H^–-DNP was monitored at 450 nm . Specific activities were calculated by using the experimentally determined extinction coefficient as described above .

Enzyme activity of the hydrolase was detected by repeated recording of UV-visible spectra between 280 and 600 nm in 1-min intervals . The reaction was performed with 50 mM Tris-HCl (pH 8.0) or 50 mM phosphate buffer (pH 8.0) containing 4 µg of hydrolase and 100 µM 2-NCH (commercially available substrate) or 2,4-DNCH (catabolic substrate obtained by the turnover of H^–-DNP by HTI and NDFR) . Specific activities with 2-NCH as substrate were determined at 340 nm with an extinction coefficient of 3,100 M^–1 cm^–1 in 50 mM phosphate buffer (pH 8.0) .

One unit of enzyme activity was defined as the amount of enzyme that converts 1 µmol of substrate per min .

Analytical methods.
Metabolites were detected and quantified at 210, 340, 390, or 420 nm by high-performance LC (HPLC) analysis (Chromeleon Chromatography Data Systems 4.38 [equipped with a UVD 170S/340S UV/Vis detector, a P 580 pump, and a Gina 50 autosampler; Dionex, Idstein, Germany]) with a Lichrospher 100 RP-18 column (Merck, Darmstadt, Germany) (250 by 4 mm; particle size, 5 µm) . The mobile phase consisted of 20% (vol/vol) methanol and 80% (vol/vol) 50 mM phosphate buffer (pH 8.0) . Proteins were removed from metabolites by filtration with pD10 desalting columns (Amersham Pharmacia) or with Amicon filters (Centricon YM-10; Millipore, Bedford, Mass.) .

LC mass spectra were obtained by negative-mode electrospray ionization (ESI^–) on an HP1100 HPLC system (Hewlett-Packard, Waldbronn, Germany) coupled to a VG Platform II Quadrupole mass spectrometer (Micromass, Manchester, United Kingdom) . Samples were resolved on a C8 reversed-phase column (Gromsil 100; Grom, Herrenberg, Germany) (125 by 4.6 mm; particle size, 5 µm) or on a Lichrosorb 100 RP-18 column (Merck) (250 by 4 mm; particle size, 5 µm) . The mobile phase consisted of 5 mM ammonium formate buffer (pH 8.0) or 20% MeOH plus 5 mM ammonium formate buffer (pH 8.0) .

Nuclear magnetic resonance (NMR) spectra of the aci-nitro and nitro forms of 2H^–-TNP (10 mM each) were recorded with an ARX 500 spectrometer (Bruker, Rheinstetten, Germany) at room temperature at a nominal frequency of 500.14 MHz (¹H) and 125.76 MHz (¹³C) for 15 s . Samples were dissolved in D₂O, and the aci-nitro and nitro forms were detected at pH 8.0 . Chemical shifts () are given in parts per million relative to tetramethylsilane as the internal standard .

Nitrite concentrations were determined spectrophotometrically by the method of Montgomery (14) .

Amino acid sequencing and sequence analysis.
The amino-terminal end of the nitrite-eliminating enzyme or the hydrolase was automatically sequenced with a 476 protein sequencing system (Applied Biosystems, Foster City, Calif.) . Database searches were performed with BLAST (1) and FASTA (17) software .

Chemicals.
2H^–-TNP and H^–-DNP were prepared according to the method of Severin et al . (22, 23) and stored at –20°C . All chemicals used were of the highest available purity and were purchased from Fluka (Taufkirchen, Germany), Merck, Roth (Karlsruhe, Germany), and Sigma-Aldrich (Taufkirchen, Germany) .

The structures of the aci-nitro and nitro forms of 2H^–-TNP were identified by ¹H NMR and ¹³C NMR spectroscopy . The NMR data of the aci-nitro form corresponded to the data of authentic 2H^–-TNP (as described before by Ebert et al.) (7) . The ¹H NMR spectrum (500 MHz, D₂O) of the nitro form showed a heptuplet at 3.74 ppm and two doublets of doublets at 3.61 and 3.52 ppm (Table 1) . This resonance pattern was similar to that of the nitro form of protonated 2H^–-TNT (24) and can be analyzed as an (AB)₂X five-spin system . The signals at 3.61 and 3.52 ppm correspond to the double set of diastereotopic methylene protons H^A and H^B at C-3 and C-5 . The geminal-coupling constant ²J (H^A, H^B) of 11.73 Hz demonstrated the presence of a doublet of doublets due to the symmetric equivalence of the diastereotopic methylene protons . The vicinal-coupling constants (4.38 and 6.53 Hz) are assigned to the methylene spectrum between H^A and H^B on one side and the H^X proton on the other side . The H^X resonance displayed a heptuplet at = 3.74 ppm .

 
The tautomers were in equilibrium, with an estimated peak area ratio of 20:80 (aci-nitro-nitro form; pH 8) calculated from the HPLC peak areas at A₃₉₀ (the UV maximum for either form) . The extinction coefficient for the aci-nitro form at pH 8.0 was 11,864 M^–1 cm^–1 . We could not calculate the extinction coefficient of the nitro form, since we did not produce it in a pure form . Therefore, we estimated the relative ratios from the peak areas for both forms detected by HPLC .

As the trisodium salt of chemically synthesized 2H^–-TNP is dissolved in buffer, the complex is instantaneously protonated to the aci-nitro form, which then slowly isomerizes to the nitro form (22, 23) . When the solution was allowed to stand at room temperature, the time needed to reach the equilibration ratio of 20:80 (aci-nitro-nitro form) was approximately 1 h at pH 7 whereas in the presence of NpdH it took approximately 0.1 min . At pH 8, the time required to reach the equilibration ratio was approximately 5 h whereas in the presence of NpdH it was approximately 1 min . Hence, the time period required for reaching the equilibrium ratio was dependent on the pH . The tautomerase catalyzed the proton-shift tautomerization, accelerating the rate 300- to 600-fold . The tautomerase is thus responsible for the proton-shift tautomerism between the two tautomeric forms .

Purification and molecular characterization of the nitrite-eliminating enzyme.
Nitrite-eliminating activity was detected in the crude extracts of both R . opacus HL PM-1 and N . simplex FJ2-1A, releasing stoichiometric amounts of nitrite from 2H^–-TNP . The enzyme was purified (as described in Materials and Methods and summarized in Table 2) from N . simplex FJ2-1A due to the easier lysis of this strain compared to the lysis of strain HL PM-1 . The specific activity of the purified protein was 57 U mg^–1 . SDS-PAGE results indicated a purity of >95% and an estimated molecular mass of 35.3 kDa . MALDI-TOF measurements gave a signal at m/z 30.50 kDa . The molecular mass of the purified nitrite-eliminating enzyme (as determined by gel filtration) was estimated to be 42 kDa . Hence, the protein is a monomer . N-terminal amino acid sequencing of the purified enzyme revealed the sequence M K N L E L A Y V G L (G) V H E X L V Y Y A A (Q/T) (H) L D (L) Y R (uncertain amino acids are in parentheses) . Comparisons to sequences in databases (GenBank and National Center for Biotechnology Information) identified no similarity to any known protein sequences .

 
Conversion of the aci-nitro form of 2H^–-TNP to H^–-DNP by the nitrite-eliminating enzyme.
To unambiguously identify the substrate and the product of nitrite elimination, the nitrite-eliminating enzyme was incubated with 2H^–-TNP and the reaction was recorded spectrophotometrically . The enzymatic turnover of 2H^–-TNP was similar to that reported before (7) . Isosbestic points at 338 and 413 nm indicated that the initial reaction mixture contained the aci-nitro form of 2H^–-TNP (pH 8.0) and that the reaction product was H^–-DNP . During the turnover, stoichiometric amounts of nitrite were released . To identify H^–-DNP, an authentic standard was prepared and identified by ¹H NMR (500 MHz, D₂O) [ (H3, H3') = 3.83 ppm (s); d (H5) = 7.49 ppm (d); d (H6) = 5.89 ppm; J (H5, H6) = 10.2 Hz] . The results corresponded to the data presented by Behrend and Heesche-Wagner (3) . HPLC analysis of H^–-DNP indicated a retention time of 1.9 min and the same UV-visible spectrum for both the standard and the product of the enzymatic conversion . Further evidence was given by HPLC-mass spectrometry (MS) analysis, revealing a single peak at an ion mass of m/z 185 which corresponded to the molecular ion [M^·]^– of H^–-DNP .

Since 2H^–-TNP exists as two tautomeric structures, we investigated whether the nitrite-eliminating enzyme possessed selectivity towards the aci-nitro or the nitro form . To show this, the final equilibrium mixture of 20:80 (aci-nitro-nitro form) served as a substrate to begin the experiment (Fig . 2A) . Addition of the nitrite-eliminating enzyme showed that only the aci-nitro form (and not the nitro form) was converted to H^–-DNP (Fig . 2B) . After removal of the nitrite-eliminating enzyme and addition of the tautomerase to solution B, the aci-nitro form developed rapidly (Fig . 2C) . Removal of the tautomerase followed by addition of the nitrite-eliminating enzyme to solution C demonstrated once more that only the aci-nitro form of 2H^–-TNP was converted to H^–-DNP (Fig . 2D) . Hence, the nitrite-eliminating enzyme uses the aci-nitro form of 2H^–-TNP as the only substrate .

 
HTI converts H^–-DNP to 2,4-DNCH.
The HTI was previously shown to hydrogenate H^–-TNP to 2H^–-TNP (8) . To show whether the analogous substance H^–-DNP additionally served as a substrate, enzyme assays were performed with H^–-DNP, F₄₂₀, NADPH, and HTI . Repetitive spectroscopic recording during the turnover of H^–-DNP by the HTI of R . opacus HL PM-1 showed a decrease in absorbance at 441 and 306 nm . A concomitant increase in absorbance at 340 nm demonstrated the generation of a new product, and HPLC analysis revealed a metabolite with a retention time of 3.2 min . The corresponding UV-visible spectrum displayed absorbance maxima at 232 and 340 nm . For confirmatory identification, the new product was analyzed by coupled HPLC-ESI^–-MS and the intense signal at m/z 187 was assigned to the molecular anion [M^·]^– of 2H^–-DNP . At pH 7.5, 2H^–-DNP is protonated to 2,4-DNCH (Fig . 1A, panel 6) . The specific activity of the HTI for H^–-DNP was 29.6 U mg^–1 .

Purification and characterization of the hydrolase converting 2,4-DNCH to 4,6-DNH.
To identify the next product and enzyme in the pathway, the activity converting the protonated form of 2H^–-DNP (i.e., 2,4-DNCH) was assayed for in crude extracts for subsequent enzyme purification . Since substrate amounts of 2,4-DNCH were unavailable by chemical or biochemical synthesis and the analogous compound 2-NCH was commercially obtainable, 2-NCH was used for further experiments . Both N . simplex FJ2-1A and R . opacus HL PM-1 grew on 2-NCH as the sole source of nitrogen . Crude extracts of both strains cultured with DNP or 2-NCH showed hydrolase activity for 2-NCH and biologically generated 2,4-DNCH . For identification, the enzyme was purified from N . simplex FJ2-1A, with 2-NCH used as the test substrate (see Materials and Methods and Table 3) . SDS-PAGE showed a single polypeptide band at 15.3 kDa with a purity of > 98% . The specific activity for 2-NCH was 24.4 U mg^–1 .

 
The molecular mass of the hydrolase was determined by MALDI-TOF measurements, giving a signal at m/z 16.989 kDa . Since the molecular mass of the purified enzyme estimated from gel filtration was 63 kDa, it can be assumed that the enzyme consists of four identical subunits . N-terminal amino acid sequencing of the purified protein by automated Edman degradation revealed the sequence M R K F W (H) V G I N V T D M D K S I E F Y R K V G F D V (S) Q (S) K (uncertain amino acids are in parentheses) . A FASTA (17) search assigned a sequence identity of 78% to the product of orfF, which encodes a putative lyase in R . opacus HL PM-1 (accession number AAK38100) (21, 25) . Hence, we propose to rename orfF as npdF .

The hydrolase was specific for 2,4-DNCH and 2-NCH and showed no activity for other metabolites (such as TNP, H^–-TNP, 2H^–-TNP, and H^–-DNP) of the catabolic pathway . Repeated UV-visible spectroscopic recording displayed a decrease in absorbance at 340 nm, indicating the disappearance of 2,4-DNCH . HPLC analysis revealed a new product with a retention time of 3.4 min . The corresponding UV-visible spectrum displayed maximum absorbance at 203 nm and a band of low intensity at 255 nm . The data were in agreement with the properties of 4,6-dinitrohexanoate (4,6-DNH) described by Lenke and Knackmuss (11) . Further confirmation of the structure was obtained by coupled HPLC-ESI^–-MS analysis . A signal at m/z 205 corresponded to the molecular anion [M^·]^– of 4,6-DNH .

Chemical and enzymatic hydrolysis of 2,4-DNCH.
To investigate chemical versus enzymatic hydrolysis of 2,4-DNCH (Fig . 1A, panel 6), UV-visible spectra of the reaction were compared under basic and acidic conditions . Hydrolysis of 2,4-DNCH (panel 6) to 4,6-DNH (panel 7) was shown to be reversible, and the equilibrium of the two forms was pH dependent . In alkaline solution (pH > 8) the reaction mixture contained only 2,4-DNCH, whereas 4,6-DNH predominated under acidic conditions (pH < 5) . Figure 3 shows the gradual spontaneous formation of 4,6-DNH from 2,4-DNCH at pH 7.5 . The extinction coefficient of 4,6-DNH was not determined, since the compound could not be prepared in a pure form; therefore, the HPLC peak areas at A₂₁₀ were calculated . In the presence of 2 µg of hydrolase, ring cleavage accelerated, showing a 15-fold increase in the peak areas corresponding to 4,6-DNH after 10 min . This shows that the hydrolase converts 2,4-DNCH to 4,6-DNH . When 4,6-DNH was incubated at pH 7.5, HPLC analysis after 30 min revealed its chemical instability as described previously (11) .

 
It has been known for more than 10 years that the polynitroaromatic chemical TNP undergoes ring reduction in R . opacus HL PM-1 and that H^–-TNP is the first metabolite (11, 20) . More recently, it became evident that related bacteria like Nocardioides spp . have the same capacity and that they use the ring reduction mechanism a second time; the resulting product is 2H^–-TNP, which serves as the substrate for nitrite release to form H^–-DNP (3, 7) . In this study, we have identified the enzymes and metabolites of TNP degradation up to ring cleavage, completing depiction of the upper TNP degradation pathway .

The present results confirm the previous suggestion that NpdH from R . opacus HL PM-1 is a tautomerase catalyzing equilibration between the nitro and the aci-nitro form of 2H^–-TNP (8) . Since nitrite elimination by the nitrite-eliminating enzyme was observed with the aci-nitro form only, this suggests that the tautomerase might be responsible for circumventing accumulation of the metabolically inert nitro form . Because tautomerization is also spontaneous, we believe that the rate of TNP degradation can be reduced in an npdH deletion mutant but not halted .

A similar pH-dependent tautomerization of the protonated dihydride Meisenheimer complex of TNT (2H^–-TNT) has been observed in 2,4,6-trinitrotoluene (TNT) dead-end metabolism in R . opacus HL PM-1 (24) . Since the strain accumulates 2H^–-TNT and is assumed to be a dead-end product (24), the tautomerase may function in TNP catabolism only . Alternatively, the nitrite-eliminating enzyme may not be able to convert 2H^–-TNT, suggesting that TNT is not utilized as a nitrogen source . Pak et al . also described the formation of tautomers of 2H^–-TNT, although they suggested that this would lead to denitration and productive degradation (16) .

The stoichiometric release and use of nitrite in TNP or DNP degradation has been demonstrated before (10, 11), and the product of nitrite release from 2H^–-TNP was shown to be H^–-DNP (3, 7) . A second source of H^–-DNP is hydrogenation of DNP, which is the first attack on DNP in DNP degradation: the HTII of R . opacus HL PM-1 and the hydride transferase of N . simplex FJ2-1A hydrogenate not only picric acid but also DNP to H^–-DNP (6, 8) . Thus, H^–-DNP is a common metabolite of the two converging pathways of TNP and DNP . It seems reasonable that these two pathways might have coevolved, since TNP and DNP are structurally very similar and occur in combination in waste streams from aniline production .

Until recently the fate of H^–-DNP was unclear . Behrend and Heesche-Wagner showed that NADPH is required for further degradation by Nocardioides sp . CB 22-2 (3) and suggested that a monooxygenolytic hydroxylation at the para position might take place, forming 2-nitrohydroquinone with release of nitrite . It was also hypothesized that H^–-DNP could be converted to 2H^–-DNP, finally giving rise to 4,6-DNH (12) . The present results confirm this: the HTI of R . opacus HL PM-1 hydrogenated H^–-DNP to 2H^–-DNP, which is protonated to form 2,4-DNCH (Fig . 1, panel 6) . The mechanism is analogous to the reduction of H^–-TNP (Fig . 1) . In the presence of NDFR and the coenzymes F₄₂₀ and NADPH, hydride is transferred to the C5 carbon atom of H^–-DNP .

Chemical hydrolysis of 2-NCH under alkaline conditions has been described by several groups as a reverse Claisen condensation (2, 13) . We showed that the hydrolytic ring opening is part of a pH-dependent equilibrium between 2,4-DNCH and 4,6-DNH . Under neutral conditions the reaction was very slow, however, such that a hydrolase should be required for rapid conversion to 4,6-DNH . In fact, crude extracts of R . opacus HL PM-1 or N . simplex FJ2-1A caused 4,6-DNH to disappear rapidly with the release of nitrite, although a product could not be identified (data not shown) . This indicates that 4,6-DNH is a true metabolite in the biodegradation of TNP and DNP; this differs from previous findings in accordance with which 4,6-DNH was suggested to be chemically formed as a minor dead-end product by spontaneous hydrolysis (11) .

In parallel with the proposed mechanism for DNP degradation described above and previously (3), Blasco et al . (4) suggested an alternative pathway of DNP degradation on the basis of detection of 3-nitroadipate in the supernatant of resting cells of Rhodococcus sp . strain RB1 . Our observations support none of these suggested mechanisms . We have provided evidence that in the upper TNP degradation pathway, three hydrogenations and ring fission take place . The bacteria appear to have evolved enzymes to cope with the highly electron-deficient aromatic ring, which obviously needs to be reduced for subsequent hydrolytic ring cleavage . This is in contrast to the general strategy of aerobic bacteria catabolizing nitroaromatic structures, i.e., oxygenolytic elimination of nitro groups and ring cleavage (15) .

We suspect that the remaining two nitro groups are cleaved from 4,6-DNH, forming carboxylic acid(s) which are funneled into the tricarboxylic acid cycle . To show this would demand radioactive labeling for detection of aliphatic compounds . Further, molecular approaches such as the creation of deletion mutants should aid in revealing the metabolites and enzymes of the lower TNP degradation pathway .

This work was supported by the German Research Foundation (DFG) .

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