Characterization of an Aldehyde Oxidoreductase From the Mesophilic Bacterium Aromatoleum aromaticum EbN1, a Member of a New Subfamily of Tungsten-Containing Enzymes

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.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1

“…Additionally, low activities of the recently characterized tungsten-containing AOR (23) were observed in all cells grown with BAm, 2-PEA, Phe, benzyl alcohol, or benzaldehyde, while it was almost absent from control cells grown with benzoate, PA, or ethylbenzene (Table 1). This corroborates the proposed function of AOR for aldehyde detoxification, because all inducing substrates are degraded via aldehyde intermediates (24).…”

The thermophilic biomass-degrading bacterium Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis

“…We also show here that GOR is part of a small minority of AOR family members that are heteromeric. These include a homolog of AOR (termed Aa AorB) that is a heterotrimer containing the equivalent of the GOR-S subunit with a string of iron-sulfur clusters (53). In addition, a second heteromeric member of the AOR family described recently is part of an enzyme complex that reduces benzoyl-CoA (54).…”

A tungsten enzyme using hydrogen as an electron donor to reduce carboxylic acids and NAD+

“…They catalyze the oxidation of various aldehydes to the respective acids and are grouped into several branches according to their sequence conservation and their substrate preference: e.g. oxidoreductases for formaldehyde (FOR) 8 , glyceraldehyde phosphate (GAPOR 9 and GOR 10 ), or wide-range spectra of different aldehydes (AOR sensu stricto [11][12][13][14][15][16] or WOR5 17 ). They are unique in biochemistry also for their ability to catalyse the thermodynamically di cult reduction of nonactivated carboxylic acids to aldehydes, which usually needs prior activation to acyl phosphates or acyl thioesters 12,15,18,19 .…”

“…oxidoreductases for formaldehyde (FOR) 8 , glyceraldehyde phosphate (GAPOR 9 and GOR 10 ), or wide-range spectra of different aldehydes (AOR sensu stricto [11][12][13][14][15][16] or WOR5 17 ). They are unique in biochemistry also for their ability to catalyse the thermodynamically di cult reduction of nonactivated carboxylic acids to aldehydes, which usually needs prior activation to acyl phosphates or acyl thioesters 12,15,18,19 . Activities of acid reduction have previously been demonstrated with AOR from Thermococcus paralvinellae 18 and Moorella thermoacetica 12,19 , albeit at much lower rates than shown for aldehyde oxidation and only when very low-potential electron donors like reduced methyl viologen or tetramethyl viologen were used as respective reductants.…”