The upper pathway of anaerobic degradation of 2-methylnaphthalene was studied with a sulphate-reducing enrichment culture, which is able to grow with naphthalene or 2-methylnaphthalene as sole carbon source and electron donor. Anaerobic degradation of 2-methylnaphthalene is initiated by an addition of fumarate to the methyl-group producing the first intermediate, naphthyl-2-methyl-succinate. In a subsequent β-oxidation of the original methyl atom, the central metabolite 2-naphthoic acid is generated. In the following pathway, the aromatic ring system is reduced, cleaved, and finally oxidised to CO2. Here, we present two new enzymatic reactions of the 2-methylnaphthalene degradation pathway that were measured in crude cell extracts. All metabolites were identified with HPLC by co-elution with synthesised reference substances. The first enzyme, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase, catalyses the activation of naphthyl-2-methyl-succinic acid to the corresponding CoA ester. The average specific activity of this enzyme was 19.6 nmol × min−1× mg of protein−1. The CoA-transfer was not inhibited by sodium borohydride and only partially by hydroxylamine, indicating that this enzyme belongs to the family III of CoA-transferases like the corresponding enzyme in the anaerobic toluene degradation pathway. The product of this CoA-transfer reaction, naphthyl-2-methyl-succinyl-CoA is then oxidised in a reaction to naphthyl-2-methylene-succinyl-CoA by the enzyme naphthyl-2-methyl-succinyl-CoA dehydrogenase. The specific activity of this enzyme was 0.115 nmol × min−1× mg of protein−1. The enzymatic activity could only be detected using phenazine methosulphate as electron acceptor. No activity was observed with natural electron acceptors such as nicotinamide adenine dinucleotide or flavin adenine dinucleotide. The two novel reactions presented here demonstrate that the original methyl-group of 2-methylnaphthalene is oxidised to the carboxyl group of 2-naphthoic acid in the upper part of the anaerobic degradation pathway.
Aromatic compounds were for a long time believed to be recalcitrant in the absence of molecular oxygen. The anaerobic degradation of aromatic hydrocarbons was demonstrated for the first time in 1985 with xylene . Since then, anaerobic degradation was shown for a large spectrum of PAHs [2, 3], but only few microbial cultures using PAH as the sole carbon source and electron donor could be obtained. For methylated aromatic compounds, the addition of fumarate to the methyl group was described as activation mechanisms [4–6]. Recently, several cultures that are able to grow with naphthalene and nitrate or sulphate as electron acceptor, were reported [7–10]. A carboxylation of naphthalene to 2-naphthoic acid was postulated as the initial activation reaction based on the identification of 2-naphthoic acid as a major metabolite and the incorporation of isotope-labelled bicarbonate into the carboxyl group of 2-naphthoic acid [7, 9]. During further metabolism, 2-naphthoic acid is subsequently reduced and undergoes a ring cleavage, as was shown on the basis of metabolites found in culture supernatants [4, 9, 11, 12]. A sulphate-reducing enrichment culture N 47 previously obtained from contaminated aquifer sediment  is able to grow on naphthalene and 2-methylnaphthalene. In supernatants of cultures fed with this substrate, naphthyl-2-methyl-succinate (NMS) and naphthyl-2-methylene-succinate (NMeS) were found , suggesting an addition of fumarate to the methyl group as the initial activating reaction. This reaction could be identified with enzyme assays in dense cell suspension . The two metabolites, NMS and NMeS, also suggested that the upper anaerobic degradation pathway of 2-methylnaphthalene occurs through an oxidation of the original methyl-atom to a carboxyl-group to form the central metabolite, 2-naphthoic acid. This sequence of reactions would be similar to the anaerobic degradation of toluene . However, the initial fumarate addition to 2-methylnaphthalene was the only enzyme reaction in the anaerobic degradation of PAHs measured in vitro so far.
Here we report on two subsequent enzymatic reactions in the anaerobic metabolism of 2-methylnaphthalene: the formation of naphthyl-2-methyl-succinyl-CoA from naphthyl-2-methyl-succinate and succinyl-CoA, and its subsequent oxidation to naphthyl-2-methylene-succinyl-CoA.
Materials and methods
Cultivation and harvest of bacteria
The naphthalene and 2-methylnaphthalene degrading sulphate-reducing culture N 47 was enriched from sediments of a tar oil-contaminated aquifer and cultivated under anoxic conditions in half-filled 120 ml serum bottles as described earlier . Naphthalene or 2-methylnaphthalene were added with a syringe as a 1.5% solution in heptamethylnonane (1 ml per bottle).
The preparation of crude cell extract was performed under strictly anoxic conditions. The cells were centrifuged for 30 min at 13,680g. In a glove box filled with N2/H2 (95/5), cell pellets were resuspended with enzyme test buffer (see below), transferred with a syringe into nitrogen-flushed vials closed with butyl rubber stoppers and broken with a french press at 137 MPa.
Synthesis of naphthyl-2-methylsuccinyl-CoA and naphthyl-2-methylenesuccinyl-CoA
Naphthyl-2-methyl-succinic acid (NMS) and naphthyl-2-methylene-succinic acid (NMeS) were synthesised as described previously . The CoA esters used in the enzymatic reactions were synthesised from internal anhydrides of NMS and NMeS. For the synthesis of internal NMS-anhydride, 50 mg of NMS was dissolved in 800 μl acetic acid at 80 °C. 50 μl acetic anhydride was added and the temperature was increased to 120 °C. After 1.5 h all acetic anhydride reacted and the acetic acid was evaporated. The remaining internal NMS-anhydride formed a yellow–brownish drop and solidified by cooling to room temperature. The pellet was dissolved in 1 ml acetonitrile. The synthesis was followed by thin layer chromatography (POLYGRAM SIL G/UV254, Machery–Nagel, Düren, Germany) and with a mixture of heptane/diethylether/acetic acid (1/1/0.1, v/v) as the running solvent.
For the synthesis of NMS–CoA, 10 mg of coenzyme A (Sigma) was dissolved in 1 ml KHCO3 (0.5 M). The NMS-anhydride solution in acetonitrile was then added in 10 aliquots of 100 μl. After each addition, the reaction mixture was shaken and incubated for 30 s. After all the NMS-anhydride solution was added, the reaction mixture was put on ice and acidified with 1 M HCl to pH 1.5. The reaction of NMS-anhydride with coenzyme A was followed by reversed phase thin layer chromatography on Alugram RP-18W/UV254 (Machery–Nagel) plates with a mixture of water/acetonitrile/acetic acid (2.5/2.5/0.1, v/v) as the running solvent.
NMeS-anhydride was synthesised analogously. As NMeS was less soluble, 2 ml of acetic acid were used to dissolve 50 mg NMeS. The synthesis of the internal anhydride was identical to the procedure described for NMS. The NMeS internal anhydride precipitated as bright yellow crystals, which were dissolved in 5 ml acetonitrile and added to the coenzyme A solution in ten aliquots of 500 μl, as described above.
Because the internal anhydrides hydrolysed spontaneously at neutral pH, a 15-fold molar excess of NMS or NMeS internal anhydride was used to synthesise the CoA ester of NMS or NMeS. To remove the remaining free acid from the acidic reaction mixture, it was extracted three times with 10 ml diethyl ether.
The stability of the synthesised CoA esters against hydrolysis was tested in 50 mM Tris–HCl buffer, pH 7.0, at room temperature. The CoA ester concentration was 40 μM. The buffer conditions and compound analysis were analogous to the succinyl-CoA:napthyl-2-methyl-succinate CoA-transferase test. The measured half-life times were 96.5 min for NMS–CoA and 5.05 min for NMeS–CoA.
The reactions of the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and of the naphthyl-2-methyl-succinyl-CoA dehydrogenase were first measured simultaneously in one assay in crude cell extracts. As the activity of the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase was measured as a backwards reaction and that of the naphthyl-2-methyl-succinyl-CoA dehydrogenase as a forward reaction, both enzymes could utilise the same substrate, NMS–CoA, producing two different products, succinyl-CoA and NMeS–CoA, respectively. To avoid any competition, NMS–CoA was added in huge excess. In more detailed studies performed in order to determine the actual electron acceptor and the influence of NaBH4 and NH2OH, the enzymatic activities were measured separately. The enzyme assays were performed at 30 °C in 50 mM Tris–HCl-buffer, pH 7.0, under strictly anoxic conditions. 1 ml cell extract was injected in a 5 ml vial flushed with N2 and closed with a butyl rubber stopper. The enzyme reaction buffer contained: Tris–HCl (50 mM), disodium succinate (2 mM), nicotinamide adenine dinucleotide (NAD+) (0.5 mM), flavin adenine dinucleotide (FAD) (0,5 mM), phenazine methosulphate (PMS) (0.5 mM), Titanium(III) citrate (100 μM) , NMS–CoA (200 μM). All stock solutions and the reaction buffer were vacuum degassed. The reaction was started by the addition of the NMS–CoA solution. To neutralise the pH shift caused by the strongly acidic NMS–CoA solution, the pH was measured with pH-indicator and adjusted with 1M NaOH. Samples of 100 μl were taken discontinuously with a Hamilton precision syringe through the stopper for 40 min. The reaction was stopped by addition of ethanol (80% final concentration).
After removal of precipitated salts by centrifugation (10 min, 15,000g), the samples were analysed by HPLC with a C18 reversed-phase column (GROM SIL 120 ODS-5 ST, 5 μm, column length 250 mm, GROM, Herrenberg, Germany) using 100 mM ammonium phosphate buffer and acetonitrile as eluents. The analysis of succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase assay occurred under the following HPLC conditions: 13 min gradient from 5% to 45% acetonitrile, 5 min isocratic at 45% acetonitrile, 1 min gradient to 5% acetonitrile, 6 min isocratic at 5% acetonitrile. The compounds were detected at 210 nm with a UV–Vis spectrometer. The observed elution times were: succinyl-CoA – 6.3 min; NMS–CoA – 13.3 min; NMS – 16.4 min.
The analysis of samples of the naphthyl-2-methyl-succinyl-CoA dehydrogenase assay was performed by isocratic elution at 40% acetonitrile. The elution times were: NMS – 7.9 min; NMeS – 8.3 min.
The protein concentrations were determined with the BioRad RC DC Protein Assay.
Succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase activity
The first enzymatic reaction in the anaerobic degradation of 2-methylnaphthalene is an addition of fumarate to the methyl group, thereby generating naphthyl-2-methyl-succinic acid. Through the analogy with anaerobic toluene degradation , we proposed that NMS becomes thereafter activated to the corresponding CoA ester for further β-oxidation to 2-naphthoic acid.
The activity of the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase was measured as the backwards reaction, i.e. the transfer of the CoA-moiety from naphthyl-2-methyl-succinyl-CoA to succinate. This was done to measure both enzymatic reactions described here simultaneously as the cell material was limited due to the extremely slow growth of the culture. The substrate NMS–CoA was added in excess to avoid a possible competition of the two measured enzymatic activities for their common substrate. As the ΔG of CoA-transfer reactions is usually close to 0, the reactions are fully reversible. The analogous enzyme in the anaerobic toluene degradation pathway was also measured as the backwards reaction .
An increase of the succinyl-CoA concentration was observed in the first 15–20 min of the experiment (Fig. 1(a)). Then, the concentration decreased because of the spontaneous hydrolysis of both, succinyl-CoA and NMS–CoA. Concomitantly, the concentration of naphthyl-2-methyl-succinic acid increased continuously. No formation of succinyl-CoA was observed in the absence of cell extract (Fig. 1(b)). Also, no transfer of the CoA-moiety from NMS–CoA to acetate or the formation of NMS–CoA in presence of NMS, CoA, and ATP could be detected. The compounds were identified with HPLC by coelution with the reference compounds. The specific activity of succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase in two independent experiments was 19.6 ± 10.6 nmol × (min × mg protein)−1 in extracts of 2-methylnaphthalene grown cells. The measured activities represent 28.5-fold the in vivo activity, which was calculated from growth experiments. This reaction could also be measured in the presence of oxygen without any significant loss of activity. In the oxic experiments, the used solutions were not degassed and no titanium(III) citrate was added.
Three different groups of CoA-transferases can be distinguished . Several inhibitors were tested to determine to which group the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase belongs to. The tests were performed according to measurements of the cinamoyl-CoA:(R)-phenyllactate CoA-transferase . The cell extract was incubated with 200 μM NMS–CoA and 10 mM NaBH4 for 10 min. Then, the reaction was started by adding succinate. No inhibition of succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase could be observed in this case. Incubation of the cell extract with 200 μM NMS–CoA for 10 min followed by 10 mM NH2OH for 20 min resulted in a residual activity of 65% compared to a control assay without inhibitor.
No activity could be observed after incubation with 200 mM NH2OH.
Naphthyl-2-methyl-succinyl-CoA dehydrogenase activity
The first oxidative reaction in the anaerobic degradation pathway of 2-methylnaphthalene was proposed to be the oxidation of naphthyl-2-methyl-succinyl-CoA to naphthyl-2-methylene-succinyl-CoA (see Fig. 3). The enzymatic activity was detected in the same assays as the one of the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase. Because of the rapid autohydrolysis of the reaction product, naphthyl-2-methylene-succinyl-CoA to NMeS and CoA (half-life time t1/2= 5 min), we measured the formation of its hydrolysis product, naphthyl-2-methylene-succinic acid (Fig. 2). A continuous formation of naphthyl-2-methylene-succinic acid was observed only in the presence of cell extract. The specific activity of the naphthyl-2-methyl-succinyl-CoA dehydrogenase in two independent experiments was 0.115 ± 0.025 nmol × (min × mg protein)−1. These activities represented 14.4% of the calculated in vivo activity. Similar to the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase, this enzyme was also insensitive to oxygen.
After the first tests, the electron acceptor of the naphthyl-2-methyl-succinyl-CoA dehydrogenase reaction was determined. No activity was observed with NAD+ or FAD. In the presence of PMS only, the activity was as high as in presence of all three electron acceptors (data not shown).
In the present study we elucidated two enzymatic reactions in the anaerobic 2-methylnaphthalene degradation pathway of the sulphate-reducing enrichment culture N 47. We have shown that naphthyl-2-methyl-succinate, which is generated by the addition of fumarate to 2-methylnaphthalene, becomes activated to the corresponding CoA-ester with succinyl-CoA as the CoA donor. This is followed by an oxidative step at the original methyl atom leading to the formation of naphthyl-2-methylene-succinyl-CoA. By these two reactions, the β-oxidation at the original methyl-atom is initiated and leads to the formation of the central metabolite 2-naphthoyl-CoA, which enters then the reductive ring cleavage pathway . The findings obtained in this study confirm the upper degradation pathway of 2-methylnaphthalene that was proposed earlier . Concerning the fact that the fumarate addition to a methyl group of an aromatic compound with a subsequent β-oxidation at the original methyl atom was detected for other substances such as toluene, xylene and cresol [6, 19], it can be postulated that this is a common reaction pattern in the anaerobic degradation of methylated aromatic hydrocarbons.
The sulphate-reducing culture N47 was transferred for at least 20 times over four years from the original inoculum, before the experiments were performed. Only one type of cells could be observed with phase contrast microscopy indicating a homogenous, although not pure culture. All attempts to obtain a pure culture failed so far, as the cells did not grow in dilutions higher than 10−4.
The investigation of catabolic pathways in PAHs-degrading anaerobic organisms is generally limited by the extremely low cell yield. The sulphate-reducing culture N47 needs for example three months to reach an OD of 0.15 and for unknown reasons, the bacteria refuse to grow in bottles larger than 150 ml. Therefore the total amount of protein that was used in this study for one enzyme assay was in the range of 1 mg. Surprisingly, the specific activities measured in the performed enzyme assays are close to those measured in studies of anaerobic toluene degradation by denitrifying bacteria .
The enzyme assays performed with the sulphate-reducing culture N47 are so far the only measured reactions of anaerobic degradation pathways of PAHs. No similar experiments were carried out with other anaerobic, 2-methylnaphthalene degrading cultures. Also, organisms or enzymes known for anaerobic toluene degradation pathways were not reported to convert two-ring aromatic hydrocarbons such as 2-methylnaphthalene or naphthalene.
The substrates used in our enzyme assays were probably mixtures of different isomers, which might have influenced the measured enzyme activities. The synthesised NMS–CoA was probably a mixture of the two CoA-esters 2-carboxymethyl-3-naphthyl-propionyl-CoA and 3-carboxy-4-naphthyl-butyryl-CoA. Only the first of these CoA-esters is proposed to be the biologically active compound . By synthesis of the analogous monoaromatic compound, benzylsuccinyl-CoA, other authors obtained a ratio of 40% 3-carboxy-4-phenylbutyryl-CoA and 60% of 2-carboxymethyl-3-phenylpropionyl-CoA . In a later publication, it was shown that purified succinyl-CoA:(R)-benzylsuccinate CoA-transferase produces only 2-carboxymethyl-3-phenylpropionyl-CoA from R-benzylsuccinate and succinyl-CoA . The naphthyl-2-methyl-succinate used in our experiments was probably also a mixture of two enantiomeres. In the anaerobic toluene degradation pathway, succinyl-CoA:(R)-benzylsuccinate CoA-transferase is inhibited by (S)-benzylsuccinyl-CoA and its enzymatic activity is lowered by 30% in the presence of racemic benzylsuccinyl-CoA . This could indicate that in the 2-methylnaphthalene degradation pathway the maximal activity of succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase is even higher than measured in the performed assays due to the inhibition through racemic NMS–CoA. The finding that the succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase was not or only partially inhibited by NaBH4 and NH2OH, indicates that the enzyme belongs to the same enzyme family(III) of CoA transferases as the analogous enzyme in the anaerobic toluene degradation pathway, i.e. succinyl-CoA:(R)-benzylsuccinate CoA-transferase.
The activity of the naphthyl-2-methyl-succinyl-CoA dehydrogenase was only detected in the presence of the artificial electron acceptor PMS and not with possible natural electron acceptors such as NAD+ or FAD. The lack of the natural electron acceptor in the enzymatic assay could have negatively influenced the measured activity of this enzyme. Also the activity of the corresponding enzyme in anaerobic toluene degradation, benzylsuccinyl-CoA dehydrogenase, was measured with artificial electron acceptors such as ferricenium hexaflourophosphate, dichlorophenol indophenol or PMS. Also in this case, no activity was observed using NAD+ or FAD .
NMS–CoA and NMeS–CoA hydrolyse spontaneously in neutral, aqueous solutions. Their half-life times (96.5 and 5.05 min, respectively), are comparable with those of the analogous CoA-thioesters from toluene degradation pathway: approximately 1 h for benzylsuccinyl-CoA and several minutes for phenylitaconyl-CoA (Johann Heider, personal communication). We have no evidences, if NMeS resulting from the spontaneous NMeS–CoA hydrolysis in vivo can be activated with CoA and re-introduced into the degradation pathway. However, other authors observed that the oxidation of benzylsuccinate in cell extracts does not take place in tests with lower protein concentrations, which was interpreted as that the short-living metabolites are passed quickly from one enzyme to another . The enzymatic oxidation of benzylsuccinyl-CoA to benzoyl-CoA was also measured in one single assay without development of detectable intermediates suggesting a fast sequence of reactions .
The reactions presented here show that the anaerobic degradation of 2-methylnaphthalene proceeds via β-oxidation on the original methyl-atom. This is analogous to the upper degradation pathway of toluene  and suggests that the fumarate molecule, which is added to the methyl group of 2-methylnaphthalene can be recycled in a later enzymatic reaction by cleavage of a succinyl-CoA moiety generating 2-naphthoyl-CoA. Other authors demonstrated also that 2-methylnaphthalene is oxidised to 2-naphthoic acid in the upper degradation pathway . However, they could not find the metabolites NMS and NMeS which can be explained by the derivatisation method for GC–MS analysis. We could detect NMS and NMeS only as methyl esters, but not as trimethylsilyl derivates, which was the derivatisation method used by other authors . The results of the present study prove also that the metabolites NMS and NMeS which were previously found in culture supernatants, are intermediates of the degradation pathway and not dead end products.
The authors thank Prof. Bernhard Schink and Prof. Stefan Haderlein for continuous support. We are especially grateful to Prof. Georg Fuchs for many fruitful discussions on this project.