bs_bs_banner 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 addi- tion 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 concentra- tion 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. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 14, 2770–2774 2772 H. Mouttaki, J. Johannes and R. U. Meckenstock 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