Information on EC 1.1.1.244 - methanol dehydrogenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.1.244
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RECOMMENDED NAME
GeneOntology No.
methanol dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
methanol + NAD+ = formaldehyde + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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redox reaction
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-
-
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reduction
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
methanol oxidation to carbon dioxide
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methanol oxidation to formaldehyde II
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Methane metabolism
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
methanol:NAD+ oxidoreductase
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CAS REGISTRY NUMBER
COMMENTARY hide
74506-37-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strains NRIC0498T, LMG1667 and NRIC0554
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strains B18LD and OH75-2a
UniProt
Manually annotated by BRENDA team
formerly Ralstonia eutropha
UniProt
Manually annotated by BRENDA team
formerly Ralstonia eutropha
UniProt
Manually annotated by BRENDA team
isolated from seawater, gene mxaF
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Manually annotated by BRENDA team
isolated from seawater, gene mxaF
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Manually annotated by BRENDA team
isolated from seawater, gene mxaF
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Manually annotated by BRENDA team
isolated from seawater, gene mxaF
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Manually annotated by BRENDA team
sp. nov., isolated from seawater, gene mxaF
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Manually annotated by BRENDA team
Methylomicrobium sp.
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Manually annotated by BRENDA team
Methylophaga spp.
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Manually annotated by BRENDA team
no activity in Acetobacter lovaniensis
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Manually annotated by BRENDA team
no activity in Acetobacter malorum
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Manually annotated by BRENDA team
no activity in Acetobacter malorum DSM 14337T
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Manually annotated by BRENDA team
no activity in Acetobacter pomorum
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Manually annotated by BRENDA team
no activity in Acetobacter pomorum DSM 11825T
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
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assimilation of methanol into central metabolism, overview
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
1,3-propandiol + NAD+
? + NADH + H+
show the reaction diagram
butanol + NAD+
butanal + NADH + H+
show the reaction diagram
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-
-
-
?
ethanol + NAD+
acetaldehyde + NADH + H+
show the reaction diagram
ethanol + NAD+
ethanal + NADH
show the reaction diagram
formaldehyde + NADH + H+
methanol + NAD+
show the reaction diagram
isopropanol + NAD+
isopropanal + NADH + H+
show the reaction diagram
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low activity
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-
?
methanol + NAD+
formaldehyde + NADH + H+
show the reaction diagram
methanol + NADP+
formaldehyde + NADPH + H+
show the reaction diagram
n-butanol + NAD+
butyraldehyde + NADH + H+
show the reaction diagram
n-butanol + NAD+
n-butanal + NADH
show the reaction diagram
n-propanol + NAD+
n-propanal + NADH
show the reaction diagram
n-propanol + NAD+
propionaldehyde + NADH + H+
show the reaction diagram
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-
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r
octanol + NAD+
octanal + NADH
show the reaction diagram
propanol + NAD+
propanal + NADH + H+
show the reaction diagram
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-
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?
propanol + NAD+
propionaldehyde + NADH + H+
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
ethanol + NAD+
ethanal + NADH
show the reaction diagram
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-
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methanol + NAD+
formaldehyde + NADH + H+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
slightly activating at 1 mM
Ni2+
activates at 1 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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reduction of aldehyde
Ca2+
slight inhibition at 1 mM
Cu2+
strong inhibition at 1 mM
iodoacetate
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weak inhibition of aldehyde reduction
N-ethylmaleimide
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weak inhibition of aldehyde reduction
Na+
slight inhibition at 1 mM
Zn2+
strong inhibition at 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Act protein
activator protein, UniProt ID I3EA59, the in vitro activity of Lysinibacillus sphaericus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase
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Activator protein
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activator protein ACT
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NudBC
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a Nudix hydrolase from Bacillus coagulans, UniProt ID G2TKZ3; a Nudix hydrolase from Bacillus coagulans, UniProt ID G2TKZ3
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NudDK
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a Nudix hydrolase from Desulfotomaculum kuznetsovii, UniProt ID F6CJI1; a Nudix hydrolase from Desulfotomaculum kuznetsovii, UniProt ID F6CJI1
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NudF protein
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can be catalytically stimulated by the Bacillus subtilis NudF protein in vitro
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NudLF
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a Nudix hydrolase from Lysinibacillus fusiformis, UniProt ID D7WXY3; a Nudix hydrolase from Lysinibacillus fusiformis, UniProt ID D7WXY3
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NudLS
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a Nudix hydrolase from Lysinibacillus sphaericus, UniProt ID B1HRL7; a Nudix hydrolase from Lysinibacillus sphaericus, UniProt ID B1HRL7
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.051 - 0.225
ethanol
1 - 7.1
formaldehyde
0.009 - 1.151
methanol
0.0072 - 0.12
n-butanol
0.02 - 2.5
NAD+
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.13 - 0.25
methanol
3.3 - 7.2
n-butanol
0.24
NAD+
pH 9.5, 30C, recombinant wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0006 - 0.0143
ethanol
0.00004 - 0.0353
methanol
0.048 - 0.903
n-butanol
0.258
NAD+
pH 9.5, 30C, recombinant wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0007
recombinant wild-type Mdh2 in Escherichia coli suspension cells, pH 7.4, 37C; recombinant wild-type Mdh in Escherichia coli suspension cells, pH 7.4, 37C, with addition of Act
0.001
recombinant wild-type Adh in Escherichia coli suspension cells, pH 7.4, 37C
0.0014
recombinant wild-type Mdh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0015
recombinant wild-type Mdh3 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0017
recombinant wild-type Mdh in Escherichia coli suspension cells, pH 7.4, 37C
0.0018
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recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0023
recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0026
recombinant wild-type Mdh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0029
recombinant wild-type Adh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0033
recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0038
recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C; recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.0051
recombinant mutant S97G Mdh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0057
recombinant mutant S97G Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0058
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recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.007
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recombinant wild-type Mdh2 in Escherichia coli suspension cells, pH 7.4, 37C
0.008 - 4
recombinant wild-type Adh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.01
recombinant wild-type Mdh1 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C
0.0114
recombinant mutant S97G Mdh2 in Escherichia coli suspension cells, pH 7.4, 37C
0.014
recombinant mutant S97G Mdh in Escherichia coli suspension cells, pH 7.4, 37C
0.0155
0.0167
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recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.021
recombinant mutant S97G Mdh in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.0213
recombinant wild-type Mdh2 in Escherichia coli suspension cells, pH 7.4, 37C, with addition of Act
0.023
recombinant wild-type Mdh3 in Escherichia coli suspension cells, pH 7.4, 37C
0.0273
recombinant wild-type Mdh3 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.03
recombinant wild-type Mdh2 in Escherichia coli suspension cells, pH 7.4, 37C
0.045
recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 7.4, 37C, with addition of Act
0.22
recombinant wild-type Mdh2 in Escherichia coli cell-free enzyme extract, pH 9.5, 45C
1.2
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enzyme activity in crude extracts of methanol grown cells, fully activated enzyme, enzyme activity is 10fold lower in the absence of activator protein
19.6
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formaldehyde reductase activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10.5
activity range, forward reaction, inactive below pH 6.0 and above pH 10.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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activity optimum at about 45C for all isozymes
60
Methylomicrobium sp.
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additional information
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thermodynamics for NAD-dependent methanol oxidation are unfavorable at low temperatures but become more favorable at higher temperatures
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4
Methylomicrobium sp.
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
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alpha, beta, 1 * 10000 + 1 * 60000
71000
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SDS-PAGE and mass spectrometry
120000
Methylomicrobium sp.
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gel filtration
363000
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ultracentrifugation sedimentation equilibrium, dissociation occurs during the centrifugation time of 80 h
430000
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calculated from electron microscopic analysis and MW of subunit
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 60
purified recombinant His-tagged enzyme, 10 min, pH 9.5, stable up to 30C, inactivation above, 13 % of activity remains after preincubation at 55C and the activity is abolished at 60C
45
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20 min, the isozymes are all stable up to; 20 min, the isozymes are stable up to, except for isozyme Mdh1, which shows about 35-40% loss of activity
50
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t1/2: above 2 min
70
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t1/2: 2 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dilution inactivates methanol dehydrogenase activity, not reductase activity, sucrose, 20% w/v, stabilizes formaldehyde reductase not methanol dehydrogenase activity, metal ions, such as Fe2+ and Zn2+, do not stabilize methanol dehydrogenase activity, glycerol does not stabilize methanol dehydrogenase activity, dithiothreitol stabilizes reductase, not methanol dehydrogenase activity, storage leads to dissociation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80 to 4C, in the presence of DTT at least 4 days stable
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4-20C, complete dissociation after 48 h
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4-20C, enzyme dissociates upon storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gel filtration
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native and recombinant proteins
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purification of activator protein
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Q-Sepharose, Phenyl-Superose, dehydrogenase activity is almost completely lost after purification, 34% reductase activity are recovered
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography; recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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wild type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli DH5alpha
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expressed in Escherichia coli, full activity only when grown in Mg2+ containing medium
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gene BFZC1_05383, recombinant expression in Escherichia coli
gene Bsph_4187, recombinant expression in Escherichia coli
gene mdh1, recombinant expression in Escherichia coli; gene mdh2, recombinant expression in Escherichia coli; gene mdh2, recombinant expression in Escherichia coli; gene mdh3, recombinant expression in Escherichia coli; gene mdh, recombinant expression in Escherichia coli; gene mdh, recombinant expression in Escherichia coli
gene mdh2 or CNE_2c13570, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain MG1655, recombinant expression of His-tagged enzyme Mdh2 in Escherichia coli strain XL-1
gene mdh2, sequence comparisons and phylogenetic tree, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3); gene mdh, sequence comparisons and phylogenetic tree, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene mxaF, DNA and amino acid sequence determination and analysis, genotypic and phylogenetic analysis and species verfication, overview
recombinant enzyme expression in Escherichia coli
three isozymes, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain ER2566
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D100N
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strongly reduced NAD+-binding, no activity
D88N
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only minor effects on activity
G13A
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only minor effects on activity
G15A
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only minor effects on activity
G95A
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impaired cofactor binding, low acitivity
K103R
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strongly reduced NAD+-binding, no activity
S101G
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site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview
S97T
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impaired cofactor binding, much higher acitivity than the wild type enzyme, no activation with ACT protein
S98G
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site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview
D38G
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site-directed mutagenesis, the mutant is active with NADP+ and NAD+ in contrast to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
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D41G
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site-directed mutagenesis, the mutant is active with NADP+ and NAD+ in contrast to the wild-type enzyme, it shows increased activity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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S101G
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site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview
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S97G
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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S98G
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site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview
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A169C
site-directed mutagenesis
A169I
site-directed mutagenesis
A169L
site-directed mutagenesis
A169M
site-directed mutagenesis
A169P
site-directed mutagenesis
A169V
from library screening, mutant CT1-2, mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type
A26V
site-directed mutagenesis, mutant CT2-2, the mutation A26V alone demolishes Mdh activity, inactive mutant
A26V/A169V
site-directed mutagenesis, mutant CT2-1, synergistic effect of mutation A26V and A169V in enzyme function increasing the activity. Mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type
A26V/A31V/A169V
site-directed mutagenesis, mutant CT4-1. Engineering of a mutant enzyme chimeric variant CT4-1 of Mdh2 that shows a 6fold higher Kcat/Km for methanol and 10fold lower Kcat/Km for n-butanol. CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation. Development of automatic high throughput screening (HTS) for Mdh evolution, overview. Mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type. CT4-1 significantly improves its methanol over C2 to C4 alcohol activity ratio compared to wild-type
A31V
from library screening, mutant CT1-1
A169L
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site-directed mutagenesis
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A169V
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from library screening, mutant CT1-2, mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type
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A26V
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site-directed mutagenesis, mutant CT2-2, the mutation A26V alone demolishes Mdh activity, inactive mutant
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A31V
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from library screening, mutant CT1-1
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additional information
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