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Literature summary extracted from

  • Good, N.M.; Vu, H.N.; Suriano, C.J.; Subuyuj, G.A.; Skovran, E.; Martinez-Gomez, N.C.
    Pyrroloquinoline quinone ethanol dehydrogenase in Methylobacterium extorquens AM1 extends lanthanide-dependent metabolism to multicarbon substrates (2016), J. Bacteriol., 198, 3109-3118 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.1.2.8 ammonium
-
 Methylorubrum extorquens
1.1.2.8 methylamine methylamine is a 5fold better activator for enzyme ExaF in vitro than ammonium Methylorubrum extorquens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.2.8 gene exaF, or exaA, DNA and amino acid sequence determination and analysis, functional recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain TOP10 Methylorubrum extorquens
1.1.2.10 expressed in Escherichia coli Top10 cells Methylorubrum extorquens

Protein Variants

EC Number Protein Variants Comment Organism
1.1.2.8 additional information a triple mutant strain (mxaF xoxF1 xoxF2, named MDH-3), deficient in the three methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, is mutated in the MDH-3 background, the quadruple mutant strain can no longer grow on methanol in minimal medium with added lanthanum (La3+) Methylorubrum extorquens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.2.10 0.0009
-
 ethanol at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 0.066
-
 formaldehyde at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 0.285
-
 acetaldehyde at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 5.98
-
 methanol at pH 9.0 and 30°C Methylorubrum extorquens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.2.8 Ca2+ ativates, active site-bound Methylorubrum extorquens
1.1.2.8 La3+ required, active site-bound, 1.3 mol of La3+ per mol of ExaF protomer, indicating a 1:1 ratio of L3+ to protomer of enzyme Methylorubrum extorquens
1.1.2.8 additional information the enzyme from strain AM1 utilizes La3+ rather than Ca2+ as a cofactor Methylorubrum extorquens
1.1.2.10 La3+ the enzyme preferentially binds La3+ over Ca2+ in the active site. 0.1 mM used in assay conditions. The enzyme contains 1.3 mol of La3+ per mol of protomer Methylorubrum extorquens

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.2.8 126400
-
 recombinant His6-tagged enzyme, gel filtration Methylorubrum extorquens
1.1.2.10 126400
-
 SEC-MALS Methylorubrum extorquens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.2.8 ethanol + 2 cytochrome c Methylorubrum extorquens
-
 acetaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 ethanol + 2 cytochrome c Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1
-
 acetaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c Methylorubrum extorquens
-
 formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1
-
 formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 additional information Methylorubrum extorquens ExaF is functionally an ethanol dehydrogenase, with secondary activities with formaldehyde, methanol, and acetaldehyde. Enzyme assays are performed with with the artificial electron acceptors phenazine methosulfate and 2,6-dichlorophenolindophenol. Methanol-independent reduction of the latter is not observed with purified enzyme ?
-
 ?
1.1.2.8 additional information Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 ExaF is functionally an ethanol dehydrogenase, with secondary activities with formaldehyde, methanol, and acetaldehyde. Enzyme assays are performed with with the artificial electron acceptors phenazine methosulfate and 2,6-dichlorophenolindophenol. Methanol-independent reduction of the latter is not observed with purified enzyme ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.2.8 Methylorubrum extorquens
-
-
-
1.1.2.8 Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1
-
-
-
1.1.2.10 Methylorubrum extorquens C5AXV8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.2.8 recombinant His6-tagged enzyme from Escherichia coli strain TOP10 by ultracentrifugation, nickel affinity chromatography, ultrafiltration, and desalting gel filtration Methylorubrum extorquens
1.1.2.10 Ni-NTA column chromatography Methylorubrum extorquens

Source Tissue

EC Number Source Tissue Comment Organism Textmining
1.1.2.8 additional information growth rate constants of Methylobacterium extorquens wild-type and mutant strains grown with ethanol as the carbon source Methylorubrum extorquens
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.2.8 ethanol + 2 cytochrome c
-
 Methylorubrum extorquens acetaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 ethanol + 2 cytochrome c
-
 Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 acetaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c
-
 Methylorubrum extorquens formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c the maximal reaction velocity of ExaF-La for formaldehyde is 23% greater than for methanol as the substrate Methylorubrum extorquens formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c
-
 Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 methanol + 2 cytochrome c the maximal reaction velocity of ExaF-La for formaldehyde is 23% greater than for methanol as the substrate Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 formaldehyde + 2 reduced cytochrome c
-
 r
1.1.2.8 additional information ExaF is functionally an ethanol dehydrogenase, with secondary activities with formaldehyde, methanol, and acetaldehyde. Enzyme assays are performed with with the artificial electron acceptors phenazine methosulfate and 2,6-dichlorophenolindophenol. Methanol-independent reduction of the latter is not observed with purified enzyme Methylorubrum extorquens ?
-
 ?
1.1.2.8 additional information ExaF is functionally an ethanol dehydrogenase, with secondary activities with formaldehyde, methanol, and acetaldehyde. Enzyme assays are performed with with the artificial electron acceptors phenazine methosulfate and 2,6-dichlorophenolindophenol. Methanol-independent reduction of the latter is not observed with purified enzyme Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 ?
-
 ?
1.1.2.10 ethanol + phenazine methosulfate highest activity Methylorubrum extorquens acetaldehyde + reduced phenazine methosulfate
-
 r
1.1.2.10 formaldehyde + reduced phenazine methosulfate the enzyme is 100times more efficient at oxidizing formaldehyde than methanol Methylorubrum extorquens methanol + phenazine methosulfate
-
 r
1.1.2.10 methanol + phenazine methosulfate
-
 Methylorubrum extorquens formaldehyde + reduced phenazine methosulfate
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.2.8 homodimer 2 * 61000, recombinant His6-tagged enzyme, SDS-PAGE Methylorubrum extorquens
1.1.2.10 homodimer x * 61000, SDS-PAGE Methylorubrum extorquens

Synonyms

EC Number Synonyms Comment Organism
1.1.2.8 exaF
-
 Methylorubrum extorquens
1.1.2.8 pyrroloquinoline quinone ethanol dehydrogenase
-
 Methylorubrum extorquens
1.1.2.10 exaF
-
 Methylorubrum extorquens
1.1.2.10 pyrroloquinoline quinone ethanol dehydrogenase
-
 Methylorubrum extorquens
1.1.2.10 quinoprotein ethanol dehydrogenase
-
 Methylorubrum extorquens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.2.8 30
-
 assay at Methylorubrum extorquens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.2.8 9
-
 assay at, alcohol oxidation Methylorubrum extorquens

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.2.8 cytochrome c
-
 Methylorubrum extorquens
1.1.2.8 additional information enzyme assays are performed with the artificial electron acceptors phenazine methosulfate and 2,6-dichlorophenolindophenol Methylorubrum extorquens
1.1.2.8 pyrroloquinoline quinone prosthetic group Methylorubrum extorquens
1.1.2.10 pyrroloquinoline quinone
-
 Methylorubrum extorquens

General Information

EC Number General Information Comment Organism
1.1.2.8 malfunction an exaF mutant is not affected for growth with ethanol Methylorubrum extorquens
1.1.2.8 additional information ExaF homology modeling using the crystal structure of the quinoprotein ethanol dehydrogenase QEDH from Pseudomonas aeruginosa, PDB 1FLG, overview. Residues E198, D317, D319, and N275 form the active site. Residue D319 might be necessary for lanthanide coordination next to catalytic aspartate D317 Methylorubrum extorquens
1.1.2.8 physiological function ExaF contributes to ethanol metabolism when La3 is present, expanding the role of lanthanides to multicarbon metabolism. ExaA quinoprotein ethanol dehydrogenase, and not the type I ADH,EC 1.1.2.4, is responsible for methanol oxidation in the MDH-3 mutant strain Methylorubrum extorquens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.2.10 2
-
 methanol at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 20
-
 acetaldehyde at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 259
-
 formaldehyde at pH 9.0 and 30°C Methylorubrum extorquens
1.1.2.10 14500
-
 ethanol at pH 9.0 and 30°C Methylorubrum extorquens