Information on EC 1.1.2.8 - alcohol dehydrogenase (cytochrome c)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.2.8
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RECOMMENDED NAME
GeneOntology No.
alcohol dehydrogenase (cytochrome c)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a primary alcohol + 2 ferricytochrome c = an aldehyde + 2 ferrocytochrome c + 2 H+
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glycolysis / Gluconeogenesis
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Chloroalkane and chloroalkene degradation
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Metabolic pathways
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Biosynthesis of secondary metabolites
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Microbial metabolism in diverse environments
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
alcohol:cytochrome c oxidoreductase
A periplasmic PQQ-containing quinoprotein. Occurs in Pseudomonas and Rhodopseudomonas. The enzyme from Pseudomonas aeruginosa uses a specific inducible cytochrome c550 as electron acceptor. Acts on a wide range of primary and secondary alcohols, but not methanol. It has a homodimeric structure [contrasting with the heterotetrameric structure of EC 1.1.2.7, methanol dehydrogenase (cytochrome c)]. It is routinely assayed with phenazine methosulfate as electron acceptor. Activity is stimulated by ammonia or amines. Like all other quinoprotein alcohol dehydrogenases it has an 8-bladed 'propeller' structure, a calcium ion bound to the PQQ in the active site and an unusual disulfide ring structure in close proximity to the PQQ.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Methylobacterium extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
butanol + 2 cytochrome c
butyraldehyde + 2 reduced cytochrome c
show the reaction diagram
diethylstilbestrol + pyrroloquinoline quinone
diethylstilbestrol 4-semiquinone + pyrroloquinoline quinol
show the reaction diagram
ethanol + 2 cytochrome c
acetaldehyde + 2 reduced cytochrome c
show the reaction diagram
ethanol + 2 cytochrome c
ethanal + 2 reduced cytochrome c
show the reaction diagram
ethanol + 2 ferricytochrome c
ethanal + 2 ferrocytochrome c
show the reaction diagram
ethanol + N,N,N',N'-tetramethyl-p-phenylenediamine
ethanal + ?
show the reaction diagram
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i.e. Wurster's Blue
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?
ethanol + oxidized 2,6-dichlorophenolindophenol
ethanal + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
methanol + 2 cytochrome c
formaldehyde + 2 reduced cytochrome c
show the reaction diagram
propanol + 2 cytochrome c
propanal + 2 reduced cytochrome c
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
butanol + 2 cytochrome c
butyraldehyde + 2 reduced cytochrome c
show the reaction diagram
diethylstilbestrol + pyrroloquinoline quinone
diethylstilbestrol 4-semiquinone + pyrroloquinoline quinol
show the reaction diagram
ethanol + 2 cytochrome c
acetaldehyde + 2 reduced cytochrome c
show the reaction diagram
ethanol + 2 ferricytochrome c
ethanal + 2 ferrocytochrome c
show the reaction diagram
methanol + 2 cytochrome c
formaldehyde + 2 reduced cytochrome c
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c
pyrroloquinoline quinone
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron
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one [2Fe-2S] cluster per enzyme
La3+
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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
Sr2+
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incubation of apo-enzyme with Sr2+ and pyrroloquinoline quinone leads to the formation of an active Sr2+-form. The Sr2+ and the Ca2+-forms of the enzyme differ in their absorption spectra. The Sr2+-form is inactivated by trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid twice as fast as the Ca2+-form.
additional information
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the enzyme from strain AM1 utilizes La3+ rather than Ca2+ as a cofactor
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ethylamine
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activating at low concentrations, inhibitory at high concentrations
pentylamine
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the alcohol oxidation activity of PpADH is strongly enhanced by pentylamine and the affinity for substrates is also improved by pentylamine as an activator, inhibitory at high concentrations
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Butylamine
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Dimethylamine
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dipropylamine
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ethanol
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does not affect the adhS gene expression but induces PQQ-ADH activity
ethylamine
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inhibitory at high concentrations
heptylamine
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Hexylamine
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Isobutylamine
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slight activation
methylamine
octylamine
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pentylamine
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the alcohol oxidation activity of PpADH is strongly enhanced by pentylamine and the affinity for substrates is also improved by pentylamine as an activator, inhibitory at high concentrations
Propylamine
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triethylamine
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trimethylamine
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tripropylamine
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additional information
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the enzyme requires ammonia or primary amines as activators in in vitro assays with artificial electron acceptors, the enzyme is activated by various primary amines, dimethylamine, and trimethylamine. tert-Butylamine is a poor activator. The hydrophobic interaction contributes to the binding of amine activators to the binding site in the enzyme
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.14 - 0.21
ethanol
0.35 - 0.43
Propanol
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24.8 - 56.6
ethanol
32.6 - 73.1
Propanol
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
115 - 395
ethanol
73 - 209
Propanol
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
112.5
ethylamine
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pH 9.0, temperature not specified in the publication
37.6
pentylamine
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pH 9.0, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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pH 9.0, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
assay at; assay at; assay at; assay at; assay at; assay at; assay at; assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.77
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sequence calculation
6.96
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sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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1 * 72000 + 1 * 45000, inactive enzyme form, SDS-PAGE; 2 * 72000 + 2 * 45000, dimer of dimers, active enzyme form, SDS-PAGE
72000
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1 * 72000 + 1 * 45000, inactive enzyme form, SDS-PAGE; 2 * 72000 + 2 * 45000, dimer of dimers, active enzyme form, SDS-PAGE
115000
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inactive enzyme form, native PAGE
120000
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inactive enzyme form, gel filtration
126400
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recombinant His6-tagged enzyme, gel filtration
136000
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gel filtration
330000
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active enzyme form, gel filtration
345000
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active enzyme form, native PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
1 * 60000 + 1 * 9000, SDS-PAGE
heterodimer
heterotetramer
homodimer
tetramer
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2 * 60000, alpha-subunit, + 2 * 9000, beta-subunit, SDS-PAGE
additional information
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enzyme interacts with a soluble cytochrome cEDH, the oxidized form being an excellent acceptor for the semiquinone form of EDH. This cytochrome is quite different from the cytochrome c551 operating in nitrate respiration
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
sequence contains a signal peptide of 34 residues
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. The amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved
diffraction to beyond 2.5 A, space group R3
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to 2.6 A resolution, by molecular replacement. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca2+ are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges contribute to different substrate specificities of these otherwise very similar enzymes. In addition to the Ca2+ in the active-site cavity, ethanol dehydrogenase contains a second Ca2+-binding site at the N-terminus, which contributes to the stability of the native enzyme
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetic acid
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme from membranes by two steps of anion exchange chormatography, hydroxyapatite chromatography, and gel filtration
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native enzyme from strain IFO3191 by ultracentrifugation and anion exchange chromatography; native enzyme from strain IFO3191 by ultracentrifugation and anion exchange chromatography; native enzyme from strain IFO3191 by ultracentrifugation and anion exchange chromatography; native enzyme from strain MSU10 by ultracentrifugation and anion exchange chromatography; native enzyme from strain MSU10 by ultracentrifugation and anion exchange chromatography; native enzyme from strain SKU1108 by ultracentrifugation and anion exchange chromatography; native enzyme from strain SKU1108 by ultracentrifugation and anion exchange chromatography; native enzyme from strain SKU1108 by ultracentrifugation and anion exchange chromatography
recombinant His6-tagged enzyme from Escherichia coli strain TOP10 by ultracentrifugation, nickel affinity chromatography, ultrafiltration, and desalting gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene adhA, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs; gene adhA, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs; gene adhB, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs; gene adhB, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs; gene adhS, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs
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
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sequence comparisons and phylogenetic tree, real-time reverse transcriptase PCR expression analysis
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ethanol does not affect the adhS gene expression but induces PQQ-ADH activity
the enzyme is induced by diethylstilbestrol, presence of diethylstilbestrol causes a 5.2fold increase in the mRNA level of the gene encoding quinoprotein alcohol dehydrogenase
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A26V
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
G55D
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
L18Q
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
T104K
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site-directed mutagenesis, the mutation leads to a complete loss of ethanol oxidizing ability
V107A
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
V36I
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
V54I
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
V70A
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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability
C105A/C106A
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mutation of residues forming a characteristic disulfide ring in the binding pocket of pyrroloquinoline quinone. Analysis by EPR spectroscopy shows that the disulfide ring is no prerequisite for the formation of the functionally important semiquinone form of pyrroloquinoline quinone
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
treatment with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid at 30C leads to an catalytically inactive apo-form. Upon incubation of the apo-form with Ca2+ and pyrroloquinoline quinone a fully active holo-enzyme is reconstituted. Incubation of apo-enzyme with Sr2+ and pyrroloquinoline quinone leads to the formation of an active Sr2+-form. The Sr2+ and the Ca2+-forms of the enzyme differ in their absorption spectra.
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
environmental protection
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