Information on EC 1.1.2.7 - methanol dehydrogenase (cytochrome c)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.2.7
-
RECOMMENDED NAME
GeneOntology No.
methanol dehydrogenase (cytochrome c)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a primary alcohol + 2 ferricytochrome cL = an aldehyde + 2 ferrocytochrome cL + 2 H+
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
1,2-dichloroethane degradation
-
-
Biosynthesis of antibiotics
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Biosynthesis of secondary metabolites
-
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Chloroalkane and chloroalkene degradation
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Glycolysis / Gluconeogenesis
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Metabolic pathways
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Methane metabolism
-
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methanol oxidation to formaldehyde I
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
methanol:cytochrome c oxidoreductase
A periplasmic quinoprotein alcohol dehydrogenase that only occurs in methylotrophic bacteria. It uses the novel specific cytochrome cL as acceptor. Acts on a wide range of primary alcohols, including ethanol, duodecanol, chloroethanol, cinnamyl alcohol, and also formaldehyde. Activity is stimulated by ammonia or methylamine. It is usually assayed with phenazine methosulfate. 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. It differs from EC 1.1.2.8, alcohol dehydrogenase (cytochrome c), in having a high affinity for methanol and in having a second essential small subunit (no known function).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Diplococcus sp.
-
-
-
Manually annotated by BRENDA team
strain A3151
-
-
Manually annotated by BRENDA team
strain PM1, gene mdh2, the organism lacks gene mxaFI, which encodes the wide-spread dimeric methanol dehydrogenase
-
-
Manually annotated by BRENDA team
P16027 (large subunit, alpha) and P14775 (small subunit, beta); strain NCIMB 9133
P16027 and P14775
UniProt
Manually annotated by BRENDA team
strain W3A1
-
-
Manually annotated by BRENDA team
strain WI 14
-
-
Manually annotated by BRENDA team
strain WI 14
-
-
Manually annotated by BRENDA team
strain FAM5, gene mdh2, the organism lacks gene mxaFI, which encodes the wide-spread dimeric methanol dehydrogenase
-
-
Manually annotated by BRENDA team
strain FAM5, gene mdh2, the organism lacks gene mxaFI, which encodes the wide-spread dimeric methanol dehydrogenase
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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
a primary alcohol + 2 ferricytochrome cL
an aldehyde + 2 ferrocytochrome cL + 2 H+
show the reaction diagram
butanol + 2,6-dichlorophenolindophenol
butyraldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
ethanol + 2,6-dichlorophenolindophenol
acetaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
heptanol + 2,6-dichlorophenolindophenol
heptaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
hexanol + 2,6-dichlorophenolindophenol
hexaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
methanol + 2 2,6-dichlorophenolindophenol
formaldehyde + 2 reduced 2,6-dichlorophenolindophenol
show the reaction diagram
methanol + 2 cytochrome cL
formaldehyde + 2 reduced cytochrome cL
show the reaction diagram
methanol + 2 ferricytochrome cL
formaldehyde + 2 ferrocytochrome cL + 2 H+
show the reaction diagram
methanol + 2,6-dichlorophenolindophenol
formaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
methanol + cytochrome cL
formaldehyde + reduced cytochrome cL
show the reaction diagram
methanol + ferricytochrome cL
formaldehyde + ferrocytochrome cL
show the reaction diagram
n-butanol + 2,6-dichlorophenolindophenol
butyraldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
n-propanol + 2,6-dichlorophenolindophenol
propionaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
octanol + 2,6-dichlorophenolindophenol
octaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
pentanol + 2,6-dichlorophenolindophenol
pentaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
propanol + 2,6-dichlorophenolindophenol
propionaldehyde + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
with phenazine methosulfate
-
-
?
sorbic alcohol + 2,6-dichlorophenolindophenol
? + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
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with phenazine methosulfate
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-
?
additional information
?
-
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
a primary alcohol + 2 ferricytochrome cL
an aldehyde + 2 ferrocytochrome cL + 2 H+
show the reaction diagram
methanol + 2 cytochrome cL
formaldehyde + 2 reduced cytochrome cL
show the reaction diagram
methanol + 2 ferricytochrome cL
formaldehyde + 2 ferrocytochrome cL + 2 H+
show the reaction diagram
methanol + cytochrome cL
formaldehyde + reduced cytochrome cL
show the reaction diagram
methanol + ferricytochrome cL
formaldehyde + ferrocytochrome cL
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,7,9-tricarboxypyrroloquinoline quinone
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PQQ, tetrahedral configuration of the C-5 atom of PQQ, configuration and binding structure, overview
cytochrome cL
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heme c
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part of cytochrome cL
phenazine methosulfate
pyrroloquinoline quinone
additional information
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an NAD(P)-independent enzyme
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
-
a cytochrome protein
Sr2+
P16027 and P14775
Ca2+ can be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+, Sr2+ shows 94% of the activity with Ca2+
additional information
-
Mg2+ cannot substitute for Ca2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
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38% inhibition at 0.1 mM
Cyclopropanol
P16027 and P14775
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Fe2+
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complete inhibition at 1 mM
phenazine methosulfate
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39% inhibition at 3 mM
additional information
-
no or poor inhibition by Ca2+, Co2+, Li+, Mg2+, Mn2+, and KCN, EDTA, and DTE
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonia
methylamine
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can replace NH4+
NH4+
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absolute requirement, NH4+ can be replaced by methylamine but not by di- or triamines
NH4Cl
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.38
butanol
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pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
0.01 - 3.58
ethanol
0.0003 - 0.105
ferricytochrome cL
0.58
Heptanol
-
pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
0.79
Hexanol
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pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
0.01 - 0.45
methanol
0.01 - 0.5
n-butanol
0.09 - 0.5
n-Propanol
0.65
Octanol
-
pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
1.34
Pentanol
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pH 9.0, 57°C, acceptor 2,6-dichlorophenolindophenol
3.69
Propanol
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pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
0.18
sorbic alcohol
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pH 9.0, 57°C, acceptor: 2,6-dichlorophenolindophenol
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15 - 0.87
ferricytochrome cL
0.11 - 4.51
methanol
additional information
additional information
Hyphomicrobium denitrificans
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kcat at different pH, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.12 - 0.7
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dependent on pH, overview
0.69
type I MDH, pH 7.0, 30°C, with cytochrome cL; type I MDH, pH 7.0, 30°C, with cytochrome cL
0.75
type II MDH, pH 7.0, 30°C, with cytochrome cL; type II MDH, pH 7.0, 30°C, with cytochrome cL
5.4
P16027 and P14775
purified enzyme
6.12
-
purified enzyme
13
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purified reconstituted Ca2+-enzyme
18
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purified native Ca2+-enzyme
18.66
type I MDH, pH 9.0, 30°C, with phenazine ethosulfate and 2,6-dichlorophenolindophenol; type I MDH, pH 9.0, 30°C, with phenazine ethosulfate and 2,6-dichlorophenolindophenol
21.03
type II MDH, pH 9.0, 30°C, with phenazine ethosulfate and 2,6-dichlorophenolindophenol; type II MDH, pH 9.0, 30°C, with phenazine ethosulfate and 2,6-dichlorophenolindophenol
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
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the enzyme oxidation step becomes rate-limiting at pH 9.0
7 - 10.5
P16027 and P14775
pH profile, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
P16027 and P14775
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
type II MDH; type II MDH
8.8
P16027 and P14775
sequence calculation
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7500
P16027 and P14775
x * 62000, alpha-subunit, + x * 7500, beta-subunit, SDS-PAGE
7580
2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, type I MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, type I MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer
8000
-
2 * 62000, alpha-subunit, + 2 * 8000, beta-subunit, alpha2beta2-structure, crystal structure determination
8500
-
2 * 66000 + 2 * 8500, alpha2beta2, crystal structure determination
27860
2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein
65980
2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, + 1 * 27860, MxaJ protein, type II MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer, and contains an additional MxaJ protein; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, type I MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer; 2 * 65980, subunit alpha, + 2 * 7580, subunit beta, type I MDH consists of two identical dimers of alpha and beta subunits organized to form the alpha2beta2 tetramer
70000
-
2 * 70000, SDS-PAGE
138000
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gel filtration
140000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
P16027 and P14775
x * 62000, alpha-subunit, + x * 7500, beta-subunit, SDS-PAGE
heterotetramer
monomer
pentamer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme or cytochrome cL, hanging drop vapour diffusion method, 16°C, 0.001 ml of 20 mg/mL enzyme or cofactor in 40 mM Tris-HCl buffer, pH 7.5, is mixed with an equal volume of precipitant colution containing 0.2 M potassium thiocyanate and 20% PEG 3350 for the enzyme crystallization, and 2 mM ZnSO4, 20% PEG 10000, and 100 mM HEPES, pH 7.3, for the crystallization of Cyt cL, 1 week to 1 month, X-ray diffraction structure determination and analysis at 1.98-2.5 A resolution; hanging-drop vapor diffusion method, X-ray structures of methanol dehydrogenase (Hd-MDH) and cytochrome cL at 2.5 A and 2.0 A resolution, respectively. Docking simulation between the coupled cytochrome cL molecules and the heterotetrameric methanol dehydrogenase
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Ba2+-containing MDH active site model to investigate the two proposed addition-elimination and hydride-transfer methanol oxidation mechanisms
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crystal structure analysis
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purified holoenzyme, hanging-drop vapour-diffusion method, 3 ml of 15 mg/ml protein solution, 20 mM Tris buffer, pH 8.0, are placed on siliconized cover slips and mixed with an equal volume of well solution, the cover slip is sealed with high-vacuum grease over a 1 ml well containing 20% PEG 8000, pH 9.0, large crystals after two weeks, X-ray diffraction structure determination and analysis at 1.2 A resolution, modeling
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X-ray diffraction structure determination
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X-ray diffraction structure determination and analysis at 1.94 A resolution
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purified native enzyme, hanging drop vapour diffusion method, 8 mg/ml protein in 25 mM Tris-HCl, pH 8.0, mixed with 0.1 M sodium cacodylate pH 6.5, 0.2 M magnesium acetate tetrahydrate and 10% v/v PEG 8000, 20°C, macrocrystals are soaked for 30 s in cryosolution consisting of crystallization solution with 20% v/v glycerol, X-ray diffraction structure determination and analysis at 1.7 A reolution, molecular replacement
purified enzyme, 5 mg/mL protein in 50 mM Tris-HCl, pH 8.25, and 13.5% PEG 8000, mixed with crystallization solution containing 1 and 50 mM methanol resulting in crystal forms A, B or C with or without incorporated methanol or ethanool, 20°C, X--ray diffraction structure determination and analysis at 1.5-3.0 A resolution, molecular modeling
X-ray diffraction strcuture determination and analysis at 2.6 A resolution, multiple isomorphous replacement, modelling
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crystal structure analysis
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crystal structure determination
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
30 min, purified enzyme, loss of 25% activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
EDTA, DTT, and Ca2+ are slightly stabilizing
P16027 and P14775
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18°C, purified native enzyme, 0.05 M sodium phosphate buffer, pH 7.5, loss of 10% of activity after 1 month
-
-80°C, purified native enzyme, 20 mM phosphate, pH 7.0, negligible loss of activity in 6 months
P16027 and P14775
4°C, purified native enzyme, 0.05 M sodium phosphate buffer, pH 7.5, loss of 30% of activity after 24 h
-
4°C, purified native enzyme, 20 mM phosphate, pH 7.0, loss of 80% activity within 1 week
P16027 and P14775
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ca2+-containing and Ca2+-free enzymes from cell-free extracts by ion exchange and hydrophobic interaction chromatography
-
native active alpha2beta2 MDH complex by ultracentrifugation, anion echange chromatgraphy, and gel filtration
native enzyme 22fold from strain AM1 in a single cation exchange chromatographical step, followed by ultrafiltration and buffer exchange, over 97% purity
P16027 and P14775
native enzyme 9fold to homogeneity by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography, followed by ultrafiltration and gel filtration
-
native isozymes by anion exchange chromatography and gel filtration, type I 7.9fold, type II 14.7fold, the lysozyme and freeze-thawing cell disruption method significantly increases the amount of type II MDH in the soluble fraction compared with strong physical disruption methods such as sonication and French Press; native isozymes by anion exchange chromatography and gel filtration, type I 7.9fold, type II 14.7fold, the lysozyme and freeze-thawing cell disruption method significantly increases the amount of type II MDH in the soluble fraction compared with strong physical disruption methods such as sonication and French Press
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis
-
gene mdh2, DNA and amino acid sequence determination and analysis, phylogenetic analysis
mxaFJGIR gene cluster encoding the large and small subunits, and protein mxaJ, organized in an operon and are transcribed as a single mRNA, DNA and amino acid sequence determination and analysis; mxaFJGIR gene cluster encoding the large and small subunits, and protein mxaJ, organized in an operon and are transcribed as a single mRNA, DNA and amino acid sequence determination and analysis
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
incubation in a calcium salt solution leads to full restoration of the Ca2+-lacking mutant enzymes to active holoenzymes, overview
P16027 and P14775
reconstitution of the active holoenzyme by incorporation of two exogenous Ca2+ into the active sites of the alpha-subunits of the alpha2beta2 tetramer, time course of Ca2+ incorporation, overview
P16027 and P14775
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