Identification of Naphthalene Carboxylase As a Prototype for The

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Environmental Microbiology (2012) 14(10), 2770–2774 doi:10.1111/j.1462-2920.2012.02768.x

Identiﬁcation of naphthalene carboxylase as a prototype for the anaerobic activation of

non-substituted aromatic hydrocarbonsemi_2768 2770..2774

Housna Mouttaki, Jörg Johannes† and aromatic hydrocarbons which are among the most Rainer U. Meckenstock* hazardous environmental contaminants. Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Introduction Germany. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of freshwater and marine sediments around Summary the globe. They are shown to be increasingly hazardous Polycyclic aromatic hydrocarbons such as naphtha- to natural habitats including aquatic life of contaminated lene are recalcitrant environmental pollutants that are sediments especially in the vicinity of high human activity only slowly metabolized by bacteria under anoxic (Neff, 2002). The US Environmental Agency has listed 16 conditions. Based on metabolite analyses of culture PAHs on the priority pollutant list. PAHs can originate from supernatants, carboxylation or methylation of naph- biogenic, pyrogenic and petrogenic sources. Because of thalene have been proposed as initial enzymatic acti- the rapid consumption of oxygen upon even low carbon vation reactions in the pathway. However, the loads, many organic-contaminated soil–water systems extremely slow growth of anaerobic naphthalene turn anoxic. Therefore, in contaminated water saturated degraders with doubling times of weeks and the little systems biodegradation has to proceed in the absence of biomass obtained from such cultures hindered the molecular oxygen. biochemical elucidation of the initial activation reac- Naphthalene is the smallest of the PAHs and is often tion, so far. Here, we provide biochemical evidence taken as a model compound for studying PAH degradation. that anaerobic naphthalene degradation is initiated It is less toxic than higher molecular mass PAHs such as via carboxylation. Crude cell extracts of the sulfate- benzo(a)pyrene, although the entire class of PAHs is of reducing enrichment culture N47 converted naphtha- great concern for environment and health (Preuss et al., lene and 13C-labelled bicarbonate to 2-[carboxyl- 2003). In studies on anaerobic naphthalene degradation, 13C]naphthoic acid at a rate of 0.12 nmol min-1 mg trace amounts of 2-naphthoic acid have been identified as protein-1. The enzyme, namely naphthalene carboxy- a metabolite in supernatants of anaerobic naphthalene- lase, catalysed a much faster exchange of 13C-labelled degrading cultures (Zhang and Young, 1997; Meckenstock bicarbonate with the carboxyl group of 2-[carboxyl- et al., 2000; Musat et al., 2009). Incorporation of 13C- -1 12C]naphthoic acid at a rate of 3.2 nmol min mg pro- labelled bicarbonate into the carboxyl group of 2-naphthoic tein-1, indicating that the rate limiting step of the acid was interpreted as a direct carboxylation of naphtha- carboxylation reaction is the activation of the naph- lene (Zhang and Young, 1997; Annweiler et al., 2000). thalene molecule rather than the carboxylation itself. Other metabolites such as naphthyl-2-methylsuccinic acid Neither the carboxylation nor the exchange reaction were also detected although these are typical intermedi- activities necessitate the presence of ATP or divalent ates of the recently elucidated 2-methylnaphthalene deg- metal ions and they were not inhibited by avidin or radation pathway (Safinowski and Meckenstock, 2006; EDTA. The new carboxylation reaction is unprec- Musat et al., 2009; Abu Laban et al., 2010; Bergmann edented in biochemistry and opens the door to under- et al., 2011a). Naphthyl-2-methylsuccinic acid is formed stand the anaerobic degradation of polycyclic when 2-methylnaphthalene is activated via fumarate addition catalysed by the glycyl radical enzyme naphthyl-2- methylsuccinate synthase (Annweiler et al., 2000; Received 30 August, 2011; revised 27 March, 2012; accepted 10 Safinowski and Meckenstock, 2004), similar to anaerobic April, 2012. *For correspondence. E-mail rainer.meckenstock@ toluene activation (Biegert et al., 1996). It was hypoth- helmholtz-muenchen.de; Tel. (+49) 89 3187 2561; Fax (+49) 89 3187 3361. †Present address: BASF, BASF SE, APD/EF, D-67117 Limburg- esized that naphthalene is first activated by methylation erhof, Germany. producing 2-methylnaphthalene, which is then further

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd Anaerobic naphthalene carboxylation to 2-naphthoic acid 2771 metabolized through the 2-methylnaphthalene degrada- dent on the amount of protein added within the concentration pathway to 2-naphthoic acid (Safinowski and Mecken- tion range of 5–50 mg of protein per assay (Fig. 1B). stock, 2006). Yet, recent studies excluded naphthalene Addition of ATP or other coenzymes or salts did not stimu- methylation and supported initial carboxylation (Musat late the reaction rate (Table 1). To confirm ATP indepen- et al., 2009). Genes encoding putative carboxylases were dence of the naphthalene carboxylase activity, a control upregulated with the aromatic hydrocarbon substrate in with N47 crude cell extract was pre-incubated with glucose benzene and naphthalene-grown cells (Abu Laban et al., and hexokinase to exclude any ATP carry over from the cell 2010; DiDonato et al., 2010; Bergmann et al., 2011b). extract. Similar enzyme activities were observed with or Here, we present conclusive biochemical evidence for the without pre-treatment. Incubation with avidin prior to sub- nature of the anaerobic enzymatic activation of naphtha- strate addition did not affect carboxylase activity indicating lene by the sulfate-reducing culture N47. that the reaction is not biotin-dependent. The enzyme reaction was inactivated by exposure to oxygen (Table 1) which could not be re-established by adding reducing Results agents like titanium(III)-citrate. Furthermore, the addition of We cultivated the sulfate-reducing naphthalene-degrading strong reducing agents such as Ti(III) citrate or sodium enrichment culture N47 in 1 l bottles, harvested cells by dithionite caused strong inhibition of the carboxylation centrifugation after 6–10 weeks of incubation, and pro- reaction, whereas the mild reductant mercaptoethanol duced crude extracts by disrupting the cells with lysozyme. inhibited only slightly (Table 1). Thus, a redox active group Soluble low-molecular-mass compounds such as ATP or appears to be essential for carboxylase activity. other coenzymes were removed via a desalting column. Unlike other carboxylases such as acetone carboxylase Earlier studies of metabolite analysis of naphthalene- or phenylphosphate carboxylase, naphthalene carboxyla- grown N47 culture, and the identification of naphthyl- tion was not dependent on the presence of divalent 2-methylsuccinic acid as a metabolite indicated a methy- cations such as Mn2+ or Mg2+ (Table 1) and addition of the lation reaction as initial step in naphthalene degradation complexing agent EDTA did not affect naphthalene car- pathway (Safinowski and Meckenstock, 2006). However, boxylase activity. The production of 2-naphthoic acid was the enzymatic formation of 2-methylnaphthalene with N47 followed by liquid chromatography/tandem mass spec- crude extract, naphthalene with S-adenosyl-L-methionine trometry (LC/MS/MS) which also allowed distinguishing or other potential methyl-donors, could not be detected between added 2-[carboxyl-12C]naphthoic acid and with crude cell extract (data not shown). In contrast, when 2-[carboxyl-13C]naphthoic acid produced by carboxylation adding naphthalene and 13C-labelled bicarbonate to the of naphthalene with [13C]bicarbonate (25 mM). When 13 assay we observed the production of C-labelled 2-[carboxyl-12C]naphthoic acid was added to the assay 2-naphthoic acid at a rate of 0.12 nmol min-1 mg-1 protein containing [13C]bicarbonate buffer, an isotope exchange (Fig. 1A) which roughly compares to the in vivo activity. No between labelled bicarbonate and the carboxyl group of 2-naphthoic acid was formed abiotically in the absence 2-naphthoic acid occurred at a specific activity of 3.2 nmol of crude cell extract. Furthermore, no additional com- min-1 mg-1 protein (Fig. 2); again this reaction was not pounds such as naphthoyl-CoA, reduced intermediates or dependent on ATP. The specific activity of the isotope naphthyl-2-methylsuccinic acid were formed during the exchange reaction was 26-fold higher than the overall incubation time. The carboxylation rate was linearly depen- carboxylation reaction indicating that the rate limiting step

Fig. 1. A. Time-course of 2-naphthoic acid production by crude extract of N47 cells with naphthalene with (ᮀ) and without ATP (5 mM) (᭡), and in the absence of crude extract ( ). The complete assay consisted of 150 mM MOPS/KOH buffer, pH 7.3, 50 ml of desalted crude extract containing 80–100 mg of crude extract protein. All data points indicate two independent replicates. B. Activity of naphthalene carboxylase determined within the ﬁrst 10 min as a function of cell extract added.

Table 1. Effects of cofactors or inhibitors on the naphthalene car- A reaction similar to naphthalene carboxylase might be boxylase activity by desalted cell extracts of N47. the carboxylation of phenol to 4-hydroxybenzoate in anaerobic phenol degradation. This carboxylation pro- Assay conditions Speciﬁc activity Relative (ﬁnal concentrations) (pmol min-1 mg-1 )a activity ceeds via formation of phenylphosphate as an intermediate, which is the true substrate for the reaction (Schühle Standard assay without cofactor 120.0 1.0 ATP (2.5 mM) 101.4 0.84 and Fuchs, 2004). The presence of a hydroxyl group

MnCl2/MgCl2 (5 mM of each) 88.9 0.74 stabilizes a negative charge on the para-carbon atom of ATP and MnCl2/MgCl2 92.8 0.78 the aromatic ring supporting an electrophilic attack by a EDTA (5 mM) 80.7 0.67 B12 (0.5 mM) 118.9 0.99 non-activated carboxyl group, as seen in the chemical TPP (0.5 mM) 107.0 0.89 Kolbe-Schmitt synthesis. As naphthalene does not Biotin 0.5 mM 99.8 0.83 contain hydroxyl groups stabilizing a carbanion and facili- Avidin 2 U 87.6 0.73 tating an electrophilic attack by carbon dioxide we can ZnCl2 (5 mM) 11.6 0.10 Sodium dithionite (1 mM) 22.3 0.19 only speculate on the mechanism of the activation reac- 2-mercaptoethanol (5 mM) 65.6 0.55 tion. Two possibilities can be envisaged, either a radical Ti(III) citrate (5 mM) 11.8 0.10 Oxygen 4.1 0.03 intermediate formation via C–H bond cleavage or an electrophilic substitution reaction on the C2 position of the a. Specific activity was calculated based on turnover rates within the naphthalene ring. first 10 min of the reaction assay as average of two independent replicates. It is generally assumed that carboxylation reactions proceed via a nucleophilic attack by a carbanion interme- of naphthalene carboxylation is the activation of naphtha- diate on the CO2 or bicarbonate to form a C–C bond lene. The isotope exchange reaction also indicates us that (O’Leary, 1992). However, we have no experimental evi- no further reaction with 2-naphthoic acid occurred in the dence indicating the formation of such naphthalene car- presence of crude cell extract. banion intermediate and if indeed it plays a role in the naphthalene carboxylase reaction. The carboxylase reaction is slightly endergonic under standard conditions with Discussion a DG0′ =+14.2 kJ mol-1 which easily allows for the forward Here, we present biochemical evidence confirming that reaction under the tested concentrations (Meckenstock naphthalene degradation proceeds via carboxylation. This and Mouttaki, 2011; Bergmann et al., 2011c). reaction has been postulated earlier based on detection of Recently, DiDonato et al. (2010) and Bergmann et al. metabolites (Zhang and Young, 1997; Meckenstock et al., (2011b) identified a carboxylase-like subunit sharing high 2000). We show here that naphthalene is probably first homology to the alpha subunit of phenylphosphate car- activated and then carboxylated to form 2-naphthoic acid boxylase of Aromatoleum aromaticum EbN1. In N47, the under strictly anaerobic conditions in the presence of N47 subunit was upregulated when grown with naphthalene crude extract. Additionally, the enzyme catalyses an compared with 2-methyl-naphthalene. This allowed the isotope exchange reaction from 2-naphthoic acid with identification of a gene cluster which products are pro- labelled bicarbonate. Interestingly, both the carboxylase posed to be involved in the activation of naphthalene via and the isotope exchange activities do necessitate neither a carboxylation reaction. Interestingly, NaphS2, another ATP nor divalent cations such as Mn2+ or Mg2+. The naph- naphthalene degrader showed a similar organization of thalene carboxylase thus differs from biotin-dependent gene cluster with gene products sharing 41% up to 82% carboxylases where the carboxyl group is activated by the biotin cofactor and where no isotope exchange reaction of the carboxyl group is occurring (Knowles, 1989). Further- more, it is surprising that such a chemically difficult reaction is catalysed without additional energy input from ATP hydrolysis. The reaction also differs from carboxylases involved in the anaerobic degradation of the aromatic hydrocarbon ethylbenzene. Ethylbenzene is initially activated by ethylbenzene dehydrogenase and further oxi- dized to acetophenone by phenylethanol dehydrogenase. Acetophenone is then carboxylated to benzoylacetate (Kniemeyer and Heider, 2001a,b; Jobst et al., 2010). The Fig. 2. Isotope exchange reaction of 2-naphthoic acid with 13 - acetophenone carboxylase reaction is ATP-dependent but H CO3 -containing buffer. 2-[carboxyl-12C]naphthoic acid (closed symbols), 2-[carboxyl-13C]naphthoic acid (open symbols) with (᭡) is proposed to proceed through an energy rich carboxy- and without (᭜) addition of ATP. ( ) indicates the control assay in phosphate intermediate. the absence of cell extract.

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 14, 2770–2774 Anaerobic naphthalene carboxylation to 2-naphthoic acid 2773 identity. Based on analogies to carboxylase-like subunits small molecules were removed using the desalting columns. identiﬁed within N47 and NaphS2 genomes, we suggest No protein was lost during the desalting step as seen in that the product of these genes are involved in the car- control experiments with lysozyme where the recovery after the desalting column treatment was about 101 Ϯ 5.6%. boxylation of naphthalene to 2-naphthoic acid. The anaerobic activation of an aromatic hydrocarbon Naphthalene activation experiment under mild conditions puts naphthalene carboxylation in line with the functionalization of non-activated alkyl C–H All experiments were carried out under strictly anoxic condi- -1 bonds (~ 410 kJ mol ) (Boll and Heider, 2010) by radical tions with N2-atmosphere in 150 mM MOPS/KOH, pH 7.3 catalysed reactions like benzylsuccinate synthase (basic buffer). Naphthalene was added as a saturated solution (Heider, 2007) or the recently described activation of (approximately 240 mM) in basic buffer. The assay was per- formed in GC microvials (approximate volume of 300 ml) crimp methane (439 kJ mol-1) in reverse methanogenesis closed with a Teﬂon coated butyl rubber septum. The tempera- (Scheller et al., 2010). The C–H bond of naphthalene is 13 ture was set to 30°C in a waterbath. A 1 M CO2 stock solution even more stable with dissociation energy of 483 kJ mol-1 was freshly prepared before each experiment by dissolving 13 for the C2 position (Lardin et al., 2001) which makes this NaH CO3 into strictly anoxic basic buffer in a crimp closed GC model reaction chemically very exciting. Intensive work vial and added immediately before addition of crude cell has been made in synthetic chemistry to functionalize one extract. Final concentrations of the tested reagents were: of the most widespread functional group, namely C–H, naphthalene, 110 mM or 2-naphthoic acid for the isotopic exchange reaction, 30 mM; ATP, 2.5 mM; hexokinase, 1 U per and form a C–C bond. The use of biocatalysts in synthetic assay; glucose, 1 mM; biotin, 0.5 mM; avidin 2 U per volume chemistry remains an attractive alternative strategy to 13 assay; NaH CO3, 25 mM; MgCl2/MnCl2, 5 mM; vitamin B12, functionalize such molecules (Koeller and Wong, 2001; 0.5 mM; 2-mercaptoethanol, 5 mM; thiamine diphosphate,

Labinger and Bercaw, 2002). 0.5 mM; Ti(III)citrate, 5 mM; ZnCl2, 5 mM; sodium dithionite, At present, there is no equivalent reaction known in 1 mM; EDTA, 5 mM; methyltetrahydrofolate, 1 mM; chemistry or biochemistry that could carboxylate hydro- S-adenosyl-L-methionine, 1 mM; methylcobalamin, 0.2 mM. carbons without energy input like ATP hydrolysis for acti- The reaction was started by adding 50 ml of crude extract from N47 cells grown with naphthalene to a final volume of 220 ml. vation. The reaction therefore constitutes a novel type of Aliquots of 50 ml were taken at the indicated time points with a carboxylation. The elucidation of the initial activation reac- N2-flushed Hamilton syringe, replacing the aliquot volume with tion in anaerobic naphthalene degradation can also be 100% N2. The reaction was stopped with the addition of 10 ml used in environmental applications to trace specific of 10% formic acid. The carboxylase activity was followed by metabolites of the pathway for assessing biodegradation measuring 13C-2-naphthoic acid formation by LC/MS/MS. in, e.g. oil reservoirs and aquifers (Aitken et al., 2004; Determination of protein content was carried out by using Jones et al., 2008). This will foster the totally unexplored the Quick Start Bradford Bye Reagent by Bio-Rad (Bio-Rad Laboratories, Hercules, CA) after boiling crude extract field of anaerobic PAH degradation as an important samples in 1 N NaOH for 10 min at 100°C giving the total process to eliminate such priority pollutants in anoxic amount of protein. The amount of protein in the assay was habitats like marine sediments or aquifers. determined by substracting the known amount of lysozyme added for cell disruption. BSA was used as a standard.

Experimental procedures LC/MS/MS Cultivation of bacteria and preparation of cell extract Metabolite analysis was carried out on an Agilent 1200 HPLC system coupled to an Applied Biosystems Q-Trap mass spec- Enrichment culture N47 was grown at 30°C in 1 l culture trometer equipped with a TurboSpray ionization source. bottles in a mineral salt medium as described by Annweiler Samples of 50 ml were separated on a Purospher Star C18e et al. (2001) with naphthalene as a carbon source and sulfate column (Merck, 125 ¥ 1.5 mm). The column oven was set to as an electron acceptor. Naphthalene was added as a 1.5% 35°C. A gradient of 25–40% acetonitrile in 0.1% acetic acid 1 w/v solution in 2,2,4,4,6,8,8-heptamethylnonane (20 ml l- over 25 min at a ﬂow rate of 0.3 ml min-1 was applied. The culture volume). Cells were harvested at mid-exponential column was washed after each run by increasing the acetoni- phase of growth and washed twice with one ml of the assay trile concentration to 90% for 1 min followed by re-equilibration buffer and then resuspended into 500 ml of the assay buffer. of the column. The ionization parameters were: declustering The cells were disrupted with 2 mg of lysozyme per ml of cell potential, -45 V, entrance potential, -4.5 V, collision energy, slurry. Cell lysis was controlled microscopically. Crude cell -14 V, collision cell exit potential, 1 V. The mass pairs detected extract was desalted through an Illustra™ NAP™-5 column in MRM mode were 171.2/127.2 for 12C-2-naphthoic acid and (GE Healthcare Europe GmbH, Freiburg, Germany) prior to 172.2/127.2 for 13C-2-naphthoic acid. the assay in order to remove small intracellular compounds. Before desalting the samples, trace amounts of non-labelled References 2-naphthoic acid (up to 30 nM) were detected in the crude extract. After desalting the samples, concentrations of Abu Laban, N., Selesi, D., Rattei, T., Tischler, P., and Meck- 2-naphthoic acid were below detection limits, indicating that enstock, R.U. (2010) Identiﬁcation of enzymes involved in

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