The biochemical properties of a new tungsten-containing aldehyde oxidoreductase from the mesophilic betaproteobacterium Aromatoleum aromaticum EbN1 (AORAa) are presented in this study. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation. AORAa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors. In contrast to previously known AORs, AORAa is a heterohexameric protein consisting of three different subunits, a large subunit containing the W-cofactor and an Fe-S cluster, a small subunit containing four Fe-S clusters, and a medium subunit containing an FAD cofactor. The presence of the expected cofactors have been confirmed by elemental analysis and spectrophotometric methods. AORAa has a pH optimum of 8.0, a temperature optimum of 40°C and is completely inactive at 50°C. Compared to archaeal AORs, AORAa is remarkably resistant against exposure to air, exhibiting a half-life time of 1 h as purified enzyme and being completely unaffected in cell extracts. Kinetic parameters of AORAa have been obtained for the oxidation of one aliphatic and two aromatic aldehydes, resulting in about twofold higher kcat values with benzyl viologen than with NAD+ as electron acceptor. Finally, we obtained evidence that AORAa is also catalyzing the reverse reaction, reduction of benzoate to benzaldehyde, albeit at very low rates and under conditions strongly favoring acid reduction, e.g., low pH and using Ti(III) citrate as electron donor of very low redox potential. AORAa appears to be a prototype of a new subfamily of bacterial AOR-like tungsten-enzymes, which differ from the previously known archaeal AORs mostly by their multi-subunit composition, their low sensitivity against oxygen, and the ability to use NAD+ as electron acceptor.
Anaerobic phenylalanine (Phe) degradation in the betaproteobacterium involves transamination and decarboxylation to phenylacetaldehyde, followed by oxidation to phenylacetate. The latter reaction is catalyzed simultaneously by two enzymes, a highly specific phenylacetaldehyde dehydrogenase (PDH) and a rather unspecific tungsten-dependent aldehyde oxidoreductase (AOR). Attempting to establish increased synthesis of AOR, we constructed a mutant lacking the gene for PDH. This mutant still grew on phenylalanine, exhibiting increased AOR activities on medium containing tungstate. In the absence of tungstate, the mutant showed initially severe growth deficiency, but resumed growth on Phe after longer incubation times. Moreover, the growth rates of the mutant increased during several re-inoculation cycles on either tungstate-proficient or -deficient media, reaching the same values as recorded in wild type strains. We confirmed AOR as the major alternative enzyme serving Phe degradation under tungstate-supplied conditions and identified and characterized the alternative NAD-dependent aldehyde dehydrogenase AldB taking over the function under tungstate-deficient conditions. Sequence analysis of the respective genes from adapted cultures under either growth condition revealed a mutation in the upstream region of the operon and a mutation within the coding region of , which are likely involved in the observed adaptation of the deletion mutant to regain fast growth on Phe. The betaproteobacterium degrades many aromatic compounds under denitrifying conditions. One of the steps of phenylalanine degradation is catalyzed by two simultaneously induced enzymes, a NAD(P)-dependent phenylacetaldehyde dehydrogenase and a W-containing aldehyde oxidoreductase. We report here that the latter fully complements a constructed deletion mutant lacking the gene for phenylacetaldehyde dehydrogenase and is overproduced after several re-inoculations. Moreover, an alternative NAD-dependent dehydrogenase is recruited to resume growth in tungstate-free medium, which does not allow the production of aldehyde oxidoreductase. This alternative enzyme is overproduced and seems to have acquired a point mutation in the active center. Our research illustrates the flexibility of environmentally important bacteria in adapting their metabolic pathways to new challenges within only a few generations.
Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their ␣-subunits, and exhibit extensive processing of their ␥-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an ␣ 2  2 ␥ 2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an ␣␥ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family. IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.
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