Information on EC 1.1.99.37 - methanol dehydrogenase (nicotinoprotein)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.99.37
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RECOMMENDED NAME
GeneOntology No.
methanol dehydrogenase (nicotinoprotein)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
methanol + acceptor = formaldehyde + reduced acceptor
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
methanol oxidation to formaldehyde III
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SYSTEMATIC NAME
IUBMB Comments
methanol:acceptor oxidoreductase
Contains Zn2+ and Mg2+. Nicotinoprotein methanol dehydrogenases have a tightly bound NADP+/NADPH cofactor that does not dissociate during the catalytic process. Instead, the cofactor is regenerated by a second substrate or electron carrier. While the in vivo electron acceptor is not known, N,N-dimethyl-4-nitrosoaniline (NDMA), which is reduced to 4-(hydroxylamino)-N,N-dimethylaniline, can serve this function in vitro. The enzyme has been detected in several Gram-positive methylotrophic bacteria, including Amycolatopsis methanolica, Rhodococcus rhodochrous and Rhodococcus erythropolis [1-3]. These enzymes are decameric, and possess a 5-fold symmetry [4]. Some of the enzymes can also dismutate formaldehyde to methanol and formate [5].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain C1
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Manually annotated by BRENDA team
strain C1
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Manually annotated by BRENDA team
strain JC1
UniProt
Manually annotated by BRENDA team
strain JC1
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ethanol + N,N-dimethyl-4-nitrosoaniline
acetaldehyde + 4-(hydroxylamino)-N,N-dimethylaniline
show the reaction diagram
formaldehyde + 4-(hydroxylamino)-N,N-dimethylaniline
methanol + N,N-dimethyl-4-nitrosoaniline
show the reaction diagram
methanol + N,N-dimethyl-4-nitrosoaniline
formaldehyde + 4-(hydroxylamino)-N,N-dimethylaniline
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
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enzyme possesses a tightly but noncovalently bound NADPH cofactor at an NADPH-to-subunit molar ratio of 0.7
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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85% inhibition at 1 mM
EDTA
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40% inhibition at 5 mM
iodoacetate
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less than 10% inhibition at a concentration of 5 mM
N-ethylmaleimide
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less than 10% inhibition at a concentration of 5 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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the low coenzyme NAD-dependent activity of MDH with C1-C4 primary alcohols is strongly stimulated by Bacillus methanolicus protein ACT, in presence of NAD(H) cofactor and Mg2+-ions. In the deduced ACT amino acid sequence, the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins is present. MDH activation by ACT involves hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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Km value of wild-type for NAD+ 0.04 mM, for NADH 0.01 mM, in absence of Mg2+, and for NAD+ 0.03 mM, for NADH 0.011 mM, in presence of Mg2+, of respectively
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.35
substrate formaldehyde
0.37
substrate ethanol
0.413
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pH 9.5, 50°C
0.48
substrate N,N-dimethyl-4-nitrosoaniline
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
363000
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or above, sedimentation equilibrium centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of quaternary protein structure by electron microscopy and image processing. Enzyme is a decameric protein displaying fivefold symmetry and possessing a tightly but noncovalently bound NADPH cofactor
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enzyme forms a decamer with 5fold symmetry, electron microscopy
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, no dissociation occurrs when the isolated protein is stored frozen
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at room temperature or at 4 °C, it dissociats into smaller fragments, probably monomers or dimers
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transcription of methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase is upregulated in cells growing on methanol compared with cells growing on glucose
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D100N
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lacks most of the bound cofactor NAD(H) and has lost all coenzyme NAD+-dependent MDH activity
D88N
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only minor differences to wild-type in kinetics
G15A
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only minor differences to wild-type in kinetics
G95A
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impaired in cofactor NAD(H) binding but retains coenzyme NAD+-dependent MDH activity; low MDH activity, 10fold decrease in affinity for NAD+/NADH, insensitive to activator protein
K103R
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lacks most of the bound cofactor NAD(H) and has lost all coenzyme NAD+-dependent MDH activity
S97G
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impaired in cofactor NAD(H) binding but retains coenzyme NAD+-dependent MDH activity; insensitive to activator protein, but displays fully activated MDH reaction rates