Abstract
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.
Highlights
Many bacteria and archaea use either molybdenum (Mo) or tungsten (W) as catalytic transition metals in enzymes catalyzing key steps of metabolism, many of which are of fundamental importance for global nutrient cycles (Hille, 2002; Hille et al, 2014)
We have reported previously that A. aromaticum EbN1 produces a W-containing aldehyde oxidoreductase (AOR) (AORAa) when grown under nitratereducing conditions with phenylalanine (Phe), which catalyzes the same reaction of the degradation pathway as a simultaneously induced phenylacetaldehyde dehydrogenase (PDH), oxidation of phenylacetaldehyde to phenylacetate (Debnar-Daumler et al, 2014; Schmitt et al, 2017)
We constructed a mutant of A. aromaticum EbN1 in which the pdh gene was deleted and replaced by a gentamicin resistance gene, leaving only AORAa to contribute to phenylacetaldehyde oxidation
Summary
Many bacteria and archaea use either molybdenum (Mo) or tungsten (W) as catalytic transition metals in enzymes catalyzing key steps of metabolism, many of which are of fundamental importance for global nutrient cycles (Hille, 2002; Hille et al, 2014). Two further tungsten-containing oxidoreductases, WOR-4 (Roy and Adams, 2002) and WOR-5 (Bevers et al, 2005) have been purified, but their physiological relevance is unknown Further orthologs of these enzymes have been characterized from various other hyperthermophilic archaeal species, such as Thermococcus litoralis (Kletzin et al, 1995), T. paralvinellae (Heider et al, 1995), Methanobacterium thermoautotrophicum (Bertram et al, 1994) or Pyrobaculum aerophilum (Hagedoorn et al, 2005). A separate branch of W-dependent enzymes of the AOR family was recently discovered in obligatory anaerobic aromatic-degrading bacteria, which were identified as benzoylCoA reductases (Kung et al, 2009) These enzymes are very large multi-subunit complexes and contain an AORtype subunit with a modified W-bis-MPT cofactor, which exhibits an additional unknown small ligand to the W atom (Weinert et al, 2015)
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