Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the environment, often introduced through anthropogenic activities like combustion of wood and fossil fuels for transport or energy production, or through leakages or spills of PAH containing substances like crude oil or coal tar. As many PAHs have toxic, carcinogenic, and / or mutagenic properties and overall adverse effects on humans and the environment, they are considered priority pollutants and their microbiological degradation in the environment is of great interest. As PAHs are recalcitrant pollutants that accumulate especially in anoxic habitats where oxygen was depleted by aerobic degradation, their anaerobic degradation is of especially relevant.
The enrichment culture N47 is able to degrade naphthalene, which is the smallest and simplest PAH and serves as a model compound, under anaerobic conditions using sulfate as electron acceptor.
Naphthalene degradation is initiated by the activation of naphthalene through carboxylation to 2-naphthoate by the naphthalene carboxylase, which is a prototype of a carboxylase targeting a non-substituted polycyclic aromatic substrate. It belongs to the UbiD-like family of (de)carboxylases, indicating that it probably utilizes a prenylated flavin mononucleotide (prFMN) co-factor. In this study, we could show that the carboxylation is a reversible two-step reaction with a stable intermediate using ATP as a co-substrate. Based on the mechanistic implications deduced from the behavior in the enzyme assays and quantum chemical calculations, we proposed a mechanism for the carboxylation reaction via an intermediate in which naphthalene is covalently bound to prFMN. First, naphthalene is bound to prFMN in a 1,3-cycloaddition, loosing aromaticity of one ring, followed by a proton abstraction leading to the stable intermediate with a single bond between naphthalene and prFMN. In the second half-reaction, an electrophilic addition of CO₂ to naphthalene C2 leads to re-establishment of the second bond between naphthalene and prFMN, forming the carboxyl group at naphthalene position 2. Then a retro-cycloaddition leads to re-aromatization of the naphthalene and cleavage of both bonds between naphthalene and prFMN.
After carboxylation, 2-naphthoate is further activated to 2-naphthoyl-CoA, which is then reduced in three consecutive two-electron reduction steps to hexahydro-2-naphthoyl-CoA. This undergoes a series of β-oxidation-like reactions leading to ring cleavage and finally pimeloyl-CoA, which is then further degraded to acetyl-CoA. In total, one molecule of naphthalene is degraded into five molecules of acetyl-CoA and one CO₂.
The acetyl-CoA is then channeled into the central metabolism, which is a novel type of chemoorganoautotrophic metabolism in N47. Earlier genomic and proteomic analysis showed that N47 possesses both genes for a complete tricarboxylic acid cycle (TCA) and Wood-
Ljungdahl pathway (WLP) and most proteins of these pathways were expressed during growth on naphthalene. In this study, we measured protein activities of both pathways and could show that both are active in N47, with activities matching the general growth rate of N47. The incorporation of isotopically labeled substrates into amino acids and fatty acids allowed to conclude which pathway was involved in their synthesis. As the building blocks for many amino acids are intermediates of the TCA cycle derived from acetyl-CoA, the amount and position of labeled carbon atoms incorporated into the amino acids can show if the acetyl-CoA they were derived from is a direct product of naphthalene degradation (fully labeled if a fully labeled naphthalene molecule was metabolized), or stems from carbon fixed from the environment (only one labeled carbon atom in acetyl-CoA if one labeled CO₂ was fixed). From these experiments we could conclude that N47 fully oxidizes naphthalene to CO₂ via the WLP, while at the same time fixing CO₂, likely through a roTCA, to generate building blocks for anabolism. This metabolism could help the cells in their extremely oligotrophic environment to prevent cell death by incomplete cell division cycles. We hypothesize that the cells first accumulate the energy necessary for cell division, likely in the form of glycogen, before actually starting the cell division cycle. Then it might happen that not enough carbon source is available in the environment to supply for the cell division, so the cells resort to CO₂ fixation to fulfill their carbon needs. This hypothesis is supported by the labeling experiments as well as the detection of glycogen in N47 that appears to be derived from CO₂.
Altogether, this study provides deeper insights into the anaerobic degradation of naphthalene, from the initial activation via carboxylation to incorporation of the final degradation products acetyl-CoA and CO₂ in the central metabolism of N47.