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Literature summary extracted from

  • Arndt, F.; Schmitt, G.; Winiarska, A.; Saft, M.; Seubert, A.; Kahnt, J.; Heider, J.
    Characterization of an aldehyde oxidoreductase from the mesophilic bacterium Aromatoleum aromaticum EbN1, a member of a new subfamily of tungsten-containing enzymes (2019), Front. Microbiol., 10, 71 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.7.5 gene ebA5355, gene cluster aorABC, DNA and amino acid sequence determination and analysis, phylogenetic analysis Aromatoleum aromaticum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.7.5 0.01
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 0.023
-
 Oxidized benzyl viologen native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum
1.2.7.5 0.063
-
 NAD+ native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum
1.2.7.5 0.067
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 0.075
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 0.082
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 0.141
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 0.155
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.7.5 Fe2+ the enzyme contains four Fe-S clusters in a small subunit Aromatoleum aromaticum
1.2.7.5 additional information an elemental analysis of purified AORAa by ICP-MS analysis reveals the presence of 1.8 W, 38 Fe, 6.2 P, and 2.3 Mg per alpha2beta2gamma2 holoenzyme, whereas no traces of Mo are detected Aromatoleum aromaticum
1.2.7.5 Tungsten in the W-cofactor Aromatoleum aromaticum

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.2.7.5 280000
-
 gel filtration Aromatoleum aromaticum

Organism

EC Number Organism UniProt Comment Textmining
1.2.7.5 Aromatoleum aromaticum Q5P0J8 EbN1 strain SR7DELTApdh
-
1.2.7.5 Aromatoleum aromaticum EbN1 Q5P0J8 EbN1 strain SR7DELTApdh
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.2.7.5 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 Aromatoleum aromaticum

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.7.5 the enzyme is purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation, purification of the native enzyme by anion exchange chromatography, desalting gel filtration, hydroxyapatite chromatography, ultrafiltration, and gel filtration Aromatoleum aromaticum

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.2.7.5 3.74
-
 purified native enzyme, pH 8.0, 28°C, substrate 4-hydroxybenzaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 4.14
-
 purified native enzyme, pH 8.0, 28°C, substrate formaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 6.11
-
 purified native enzyme, pH 8.0, 28°C, substrate glutardialdehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 6.58
-
 purified native enzyme, pH 8.0, 28°C, substrate 2-aminobenzaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 8.95
-
 purified native enzyme, pH 8.0, 28°C, substrate crotonaldehyde, with NAD+ Aromatoleum aromaticum
1.2.7.5 9.6
-
 purified native enzyme, pH 8.0, 28°C, substrate acetaldehyde, with NAD+ Aromatoleum aromaticum
1.2.7.5 10.3
-
 purified native enzyme, pH 8.0, 28°C, substrate glyceraldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 11.2
-
 purified native enzyme, pH 8.0, 28°C, substrate phenylacetaldehyde, with NAD+ Aromatoleum aromaticum
1.2.7.5 12
-
 purified native enzyme, pH 8.0, 28°C, substrate benzaldehyde, with NAD+ Aromatoleum aromaticum
1.2.7.5 12
-
 purified native enzyme, pH 8.0, 28°C, substrate crotonaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 12.3
-
 purified native enzyme, pH 8.0, 28°C, substrate propionaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 19.9
-
 purified native enzyme, pH 8.0, 28°C, substrate acetaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 23.6
-
 purified native enzyme, pH 8.0, 28°C, substrate benzaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 25.1
-
 purified native enzyme, pH 8.0, 28°C, substrate phenylacetaldehyde, with oxidized benzyl viologen Aromatoleum aromaticum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.7.5 2-aminobenzaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum 2-aminobenzoate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 2-aminobenzaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum EbN1 2-aminobenzoate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 4-hydroxybenzaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum 4-hydroxybenzoate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 4-hydroxybenzaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum EbN1 4-hydroxybenzoate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 acetaldehyde + H2O + 2 NAD+
-
 Aromatoleum aromaticum acetate + 2 NADH + 2 H+
-
 ?
1.2.7.5 acetaldehyde + H2O + 2 NAD+
-
 Aromatoleum aromaticum EbN1 acetate + 2 NADH + 2 H+
-
 ?
1.2.7.5 acetaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum acetate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 acetaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum EbN1 acetate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 benzaldehyde + H2O + 2 FAD
-
 Aromatoleum aromaticum benzoate + 2 FADH2
-
 ?
1.2.7.5 benzaldehyde + H2O + 2 NAD+
-
 Aromatoleum aromaticum benzoate + 2 NADH + 2 H+
-
 ?
1.2.7.5 benzaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum benzoate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 benzoate + Ti(III) citrate + 2 H+
-
 Aromatoleum aromaticum benzaldehyde + Ti(I) citrate + H2O
-
 ?
1.2.7.5 crotonaldehyde + H2O + 2 NAD+
-
 Aromatoleum aromaticum crotonate + 2 NADH + 2 H+
-
 ?
1.2.7.5 crotonaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum crotonate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 formaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum formate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 glutardialdehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum ?
-
 ?
1.2.7.5 glyceraldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum glycerate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 additional information enzyme AORAa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors, about twofold higher kcat values with benzyl viologen than with NAD+ as electron acceptor. Enzyme 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. Substrate specificity, overview Aromatoleum aromaticum ?
-
-
1.2.7.5 additional information enzyme AORAa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors, about twofold higher kcat values with benzyl viologen than with NAD+ as electron acceptor. Enzyme 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. Substrate specificity, overview Aromatoleum aromaticum EbN1 ?
-
-
1.2.7.5 phenylacetaldehyde + H2O + 2 NAD+
-
 Aromatoleum aromaticum phenylacetate + 2 NADH + 2 H+
-
 ?
1.2.7.5 phenylacetaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum phenylacetate + 2 H+ + 2 reduced benzyl viologen
-
 ?
1.2.7.5 propionaldehyde + H2O + 2 oxidized benzyl viologen
-
 Aromatoleum aromaticum propionate + 2 H+ + 2 reduced benzyl viologen
-
 ?

Subunits

EC Number Subunits Comment Organism
1.2.7.5 heterohexamer alpha2beta2gamma2 composition of the enzyme Aromatoleum aromaticum
1.2.7.5 More 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 Aromatoleum aromaticum

Synonyms

EC Number Synonyms Comment Organism
1.2.7.5 aldehyde oxidoreductase
-
 Aromatoleum aromaticum
1.2.7.5 aorA
-
 Aromatoleum aromaticum
1.2.7.5 AORAa
-
 Aromatoleum aromaticum
1.2.7.5 ebA5355
-
 Aromatoleum aromaticum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.7.5 40
-
-
 Aromatoleum aromaticum

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.2.7.5 additional information
-
 completely inactive at 50°C Aromatoleum aromaticum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.7.5 35
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 40
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 43
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 54
-
 NAD+ native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum
1.2.7.5 81
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 91
-
 Oxidized benzyl viologen native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum
1.2.7.5 92
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 103
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.7.5 8
-
-
 Aromatoleum aromaticum

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.7.5 FAD in a medium size subunit, encoded by gene aorC Aromatoleum aromaticum
1.2.7.5 Fe-S cluster the enzyme contains four Fe-S clusters in a small subunit encoded by gene aorA Aromatoleum aromaticum
1.2.7.5 additional information a tungsten-cofactor encoded by gene aorB Aromatoleum aromaticum
1.2.7.5 NAD+
-
 Aromatoleum aromaticum

Expression

EC Number Organism Comment Expression
1.2.7.5 Aromatoleum aromaticum the enzyme is induced during anaerobic growth on phenylalanine (Phe) and a number of other substrates up

General Information

EC Number General Information Comment Organism
1.2.7.5 evolution the aldehyde oxidoreductase belongs to the subfamily of tungsten-containing enzymes. AORAa appears to be a prototype of a distinct 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. Phylogenetic tree of the AOR enzyme family, overview Aromatoleum aromaticum
1.2.7.5 additional information 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 Aromatoleum aromaticum

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.2.7.5 277.4
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 533.3
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 730.5
-
 acetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 857.1
-
 NAD+ native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum
1.2.7.5 987.8
-
 phenylacetaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 1373
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with oxidized benzyl viologen Aromatoleum aromaticum
1.2.7.5 3500
-
 benzaldehyde native enzyme, pH 8.0, 28°C, with NAD+ Aromatoleum aromaticum
1.2.7.5 3946.5
-
 Oxidized benzyl viologen native enzyme, pH 8.0, 28°C, with benzaldehyde Aromatoleum aromaticum