Information on EC 1.1.3.38 - vanillyl-alcohol oxidase

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The expected taxonomic range for this enzyme is: Penicillium simplicissimum

EC NUMBER
COMMENTARY hide
1.1.3.38
-
RECOMMENDED NAME
GeneOntology No.
vanillyl-alcohol oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Vanillyl alcohol + O2 = vanillin + H2O2
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminobenzoate degradation
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
vanillyl alcohol:oxygen oxidoreductase
Vanillyl-alcohol oxidase from Penicillium simplicissimum contains covalently bound FAD. It converts a wide range of 4-hydroxybenzyl alcohols and 4-hydroxybenzylamines into the corresponding aldehydes. The allyl group of 4-allylphenols is also converted into the -CH=CH-CH2OH group.
CAS REGISTRY NUMBER
COMMENTARY hide
143929-24-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-1-(4'-Hydroxyphenyl)ethanol + O2
?
show the reaction diagram
-
-
-
-
-
(R,S)-1-(4'-Hydroxyphenyl)propanol + O2
?
show the reaction diagram
-
-
-
-
-
(S)-1-(4'-Hydroxyphenyl)ethanol + O2
?
show the reaction diagram
-
-
-
-
-
1-(4'-Hydroxyphenyl)-2-butanone + O2
?
show the reaction diagram
-
-
-
-
-
2-(4'-Hydroxyphenyl)ethanol + O2
?
show the reaction diagram
-
-
-
-
-
2-amino-p-cresol + O2
?
show the reaction diagram
-
-
-
-
?
2-Methoxy-4-n-propylphenol + O2
1-(4'-Hydroxy-3'-methoxyphenyl)propanol
show the reaction diagram
2-methyl-p-cresol + O2
?
show the reaction diagram
-
-
-
-
?
3-(4'-Hydroxyphenyl)propanol + O2
?
show the reaction diagram
-
-
-
-
-
3-methoxy-4-hydroxybenzyl alcohol + O2
3-methoxy-4-hydroxybenzaldehyde
show the reaction diagram
-
i.e. vanillyl alcohol
i.e. vanillin
-
?
4-(3'-Methylcrotyl)phenol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene
show the reaction diagram
4-(methoxymethyl)phenol + O2
1-(4'-hydroxyphenyl)ethanol + H2O2
show the reaction diagram
-
-
like native enzyme the mutant enzymes T457E, D170A and d170S preferentially form the (R)-enantiomer. The mutant enzymes D170A/T457E and D170S/T457E exhibit an inverted stereospecificity
-
?
4-(Methoxymethyl)phenol + O2
4-Hydroxybenzaldehyde
show the reaction diagram
4-(methoxymethyl)phenol + O2
?
show the reaction diagram
4-butylphenol + O2
4-but-1-en-1-ylphenol + 4-[(1R)-1-hydroxybutyl]phenol
show the reaction diagram
-
-
mutant D170E: 0% alcohol + 100% alkene. Mutant D170S: 82% alcohol + 18% alkene
-
?
4-ethylphenol + O2
(R)-1-(4'-hydroxyphenyl)ethanol
show the reaction diagram
-
-
product formed by wild-type enzyme and mutant enzymes D170S and D170A.D170A/T457E and D170S/T457E form (S)-1-(4'-hydroxyphenyl)ethanol. The hydroxylation reaction in D170S is nearly completely blocked and this mutant converts short-chain alkylphenols to the corresponding alkenes. Mutant D170E: 8% alcohol + 92% alkene. Mutant D170S: 92% alcohol + 8% alkene
-
?
4-ethylphenol + O2
(R)-1-(4'-hydroxyphenyl)ethanol + 4-vinylphenol
show the reaction diagram
-
-
product formed by wild-type enzyme and mutant enzyme D170S. The hydroxylation reaction in D170S is nearly completely blocked and this mutant converts short-chain alkylphenols to the corresponding alkenes. Mutant D170E: 8% alcohol + 92% alkene. Mutant D170S: 92% alcohol + 8% alkene
-
?
4-Ethylphenol + O2
1-(4'-Hydroxyphenyl)ethanol + 4-vinylphenol + 4-hydroxyacetophenone
show the reaction diagram
4-Hydroxy-3-methoxybenzylamine + O2
?
show the reaction diagram
-
i.e. vanillylamine
-
-
-
4-Hydroxy-3-methoxyphenylglycol + O2
?
show the reaction diagram
-
-
-
-
-
4-hydroxybenzyl alcohol + O2
4-hydroxybenzaldehyde + H2O2
show the reaction diagram
-
at 75% of the activity with vanillyl alcohol
-
-
4-Isopropylphenol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene
show the reaction diagram
-
-
20% 4-hydroxyphenylalcohol + 80% 4-hydroxyphenylalkene
-
4-isopropylphenol + O2
4-isopropenylphenol + ?
show the reaction diagram
-
-
mutant D170E: 8% alcohol + 92% alkene. Mutant D170S: 80% alcohol + 20% alkene
-
?
4-Methylphenol + O2
?
show the reaction diagram
-
transient intermediate: p-quinone methide of the aromatic substrate in complex with reduced enzyme. Ordered sequential binding mechanism in which the rate of flavin reduction determines the turnover rate while the reduced enzyme-p-quinone methide binary complex rapidly reacts with dioxygen
-
-
-
4-n-Butylphenol + O2
4-Hydroxyphenylalkene
show the reaction diagram
-
-
93% cis-4-hydroxyphenylalkene + 7% trans-4-hydroxyphenylalkene
-
4-n-Heptylphenol + O2
4-Hydroxyphenylalkene
show the reaction diagram
-
-
50% cis-4-hydroxyphenylalkene + 50% trans-4-hydroxyphenylalkene
-
4-n-Pentylphenol + O2
4-Hydroxyphenylalkene
show the reaction diagram
4-n-propylphenol + O2
1-(4'-hydroxyphenyl)propanol + 4-(prop-1-en-1-yl)phenol
show the reaction diagram
-
-
mutant D170E: 7% alcohol + 93% alkene. Mutant D170S: 96% alcohol + 4% alkene
-
?
4-propylphenol + O2
(R)-1-(4'-hydroxyphenyl)propanol + cis-1-(4'-hydroxyphenyl)propene + trans-1-(4'-hydroxyphenyl)propene
show the reaction diagram
-
-
in an aqueous medium the enzyme produces mainly (R)-1-(4'-hydroxyphenyl)propanol and low but equal amounts of cis-1-(4'-hydroxyphenyl)propene and trans-1-(4'-hydroxyphenyl)propene. In acetonitrile or toluene the concentration of the alcohol product decreases and the concentration of the cis-alkene product, but not the trans-alkene product increases
-
?
4-Propylphenol + O2
1-(4'-Hydroxyphenyl)propanol + 4-propenylphenol + 1-(4'-hydroxyphenyl)propanone
show the reaction diagram
4-sec-Butylphenol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene
show the reaction diagram
-
-
26% 4-hydroxyphenylalcohol + 74% 4-hydroxyphenylalkene. 100% cis-4-hydroxyphenylalkene
-
4-sec-butylphenol + O2
?
show the reaction diagram
-
-
mutant D170E: 1% alcohol + 99% alkene. Mutant D170S: 78% alcohol + 22% alkene
-
?
5,6,7,8-Tetrahydro-2-naphthol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene + 4-hydroxyphenylalkanone
show the reaction diagram
-
-
4% 4-hydroxyphenylalcohol + 94% 4-hydroxyphenylalkene + 2% 4-hydroxyphenylalkanone
-
5-Indanol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene + 4-hydroxyphenylalkanone
show the reaction diagram
-
-
16% 4-hydroxyphenylalcohol + 24% 4-hydroxyphenylalkene + 60% 4-hydroxyphenylalkanone
-
chavicol + O2
coumaryl alcohol
show the reaction diagram
-
-
-
-
creosol + O2
?
show the reaction diagram
-
-
-
-
?
creosol + O2
vanillin + ?
show the reaction diagram
-
the conversion of cresol proceeds via a two-step enzymatic process. In the first step creosol is hydroxylated to yield vanillyl alcohol, and in the second step vanillyl alcohol is oxidized to yield vanillin
-
-
?
eugenol + O2
?
show the reaction diagram
-
-
-
-
?
eugenol + O2
coniferyl alcohol + H2O
show the reaction diagram
eugenol + O2
coniferyl alcohol + H2O2
show the reaction diagram
Metanephrine + O2
?
show the reaction diagram
-
-
-
-
-
Norepinephrine + O2
?
show the reaction diagram
-
-
-
-
-
Normetanephrine + O2
?
show the reaction diagram
-
-
-
-
-
p-cresol + O2
?
show the reaction diagram
-
-
-
-
?
vanillyl alcohol + O2
?
show the reaction diagram
-
-
-
-
?
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
vanillyl alcohol + phenazine methosulfate
vanillin + reduced phenazine methosulfate
show the reaction diagram
-
-
-
-
vanillylamine + O2
?
show the reaction diagram
-
-
-
-
?
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
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8alpha-(N3-Histidyl)-FAD
flavin
-
a covalent 5-(4'-hydroxybenzyl)-FAD adduct is formed during reaction with 4-methylphenol
additional information
-
no activity with riboflavin, FMN, ADP, or AMP
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-Nitro-p-cresol
-
-
4-(1-Heptenyl)-phenol
-
the crystal structure of the enzyme in complex with the inhibitor shows that the catalytic cavity is completely filled by the inhibitor
4-hydroxyphenylethyl alcohol
-
-
4-vinylphenol
-
-
cinnamyl alcohol
-
strong competitive inhibitor of vanillyl-alcohol oxidation
Medium-chain 4-alkylphenol
-
product inhibition
-
p-mercuribenzoate
-
one Cys residue reacts rapidly without loss of enzyme activity, three sulfhydryl groups then react in an 'all or none process' involving enzyme inactivation and dissociation of the octamer into dimers, antichaotropic salts protect from mercuration
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.222
(R)-1-(4'-hydroxyphenol)ethanol
-
-
0.03
(R,S)-1-(4'-Hydroxyphenyl)propanol
-
-
0.026
(S)-1-(4'-Hydroxyphenyl)ethanol
-
-
0.128
1-(4'-hydroxyphenol)-2-butanone
-
-
0.1
2-(4'-hydroxyphenyl)ethanol
-
-
0.029 - 0.254
2-amino-p-cresol
0.006
2-Methoxy-4-n-propylphenol
-
-
0.001 - 0.021
2-methyl-p-cresol
0.065
4-(3'-Methylcrotyl)phenol
-
-
0.004 - 238
4-(Methoxymethyl)phenol
0.006
4-butylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.007 - 0.048
4-Ethylphenol
0.24
4-Hydroxy-3-methoxybenzylamine
-
-
0.016 - 0.088
4-Isopropylphenol
0.031
4-Methylphenol
-
-
0.002
4-n-Butylphenol
-
-
0.008
4-n-Pentylphenol
-
-
0.01
4-n-Propylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.003 - 0.004
4-Propylphenol
0.062 - 0.075
4-sec-Butylphenol
0.094
5,6,7,8-Tetrahydro-2-naphthol
-
-
0.077
5-Indanol
-
-
0.0048
chavicol
-
-
0.001 - 0.05
creosol
0.0002 - 0.019
Eugenol
1.6
metanephrine
-
-
0.042
n-heptylphenol
-
-
-
2.9
norepinephrine
-
-
1.4
normetanephrine
-
-
0.031
p-Cresol
-
pH 7.5, 25C, wild-type enzyme
0.043
phenazine methosulfate
-
-
0.0037
propylphenol
-
-
-
0.006 - 0.73
Vanillyl alcohol
0.048
vanillylamine
-
pH 7.4, 25C
additional information
additional information
-
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.7
(R)-1-(4'-hydroxyphenol)ethanol
Penicillium simplicissimum
-
-
3
(R,S)-1-(4'-Hydroxyphenyl)propanol
Penicillium simplicissimum
-
-
4.4
(S)-1-(4'-Hydroxyphenyl)ethanol
Penicillium simplicissimum
-
-
0.3
1-(4'-hydroxyphenol)-2-butanone
Penicillium simplicissimum
-
-
0.004
2-(4'-hydroxyphenyl)ethanol
Penicillium simplicissimum
-
-
0.001 - 0.099
2-amino-p-cresol
3.2
2-Methoxy-4-n-propylphenol
Penicillium simplicissimum
-
-
4.8
2-methoxy-4-propylphenol
Penicillium simplicissimum
-
-
0.002 - 0.063
2-methyl-p-cresol
0.1
3-(4'-hydroxyphenol)propanol
Penicillium simplicissimum
-
-
1.4
4-(3'-Methylcrotyl)phenol
Penicillium simplicissimum
-
-
5.3
4-(methoxymethyl)-phenol
Penicillium simplicissimum
-
-
0.002 - 3.9
4-(Methoxymethyl)phenol
0.0001 - 0.12
4-butylphenol
0.17 - 2.5
4-Ethylphenol
1.3
4-Hydroxy-3-methoxybenzylamine
Penicillium simplicissimum
-
-
3.6
4-hydroxy-3-methoxyphenylglycol
Penicillium simplicissimum
-
-
0.13 - 1.3
4-Isopropylphenol
0.005
4-Methylphenol
Penicillium simplicissimum
-
-
1.2
4-n-Butylphenol
Penicillium simplicissimum
-
-
0.3
4-n-Pentylphenol
Penicillium simplicissimum
-
-
0.0002 - 4.2
4-n-Propylphenol
3.9
4-Propylphenol
Penicillium simplicissimum
-
-
0.05 - 0.5
4-sec-Butylphenol
0.7
5,6,7,8-Tetrahydro-2-naphthol
Penicillium simplicissimum
-
-
0.5
5-Indanol
Penicillium simplicissimum
-
-
6.5
chavicol
Penicillium simplicissimum
-
-
0.003 - 0.17
creosol
2.7
ethylphenol
Penicillium simplicissimum
-
-
0.01 - 39
Eugenol
0.8
metanephrine
Penicillium simplicissimum
-
-
0.3
norepinephrine
Penicillium simplicissimum
-
-
0.7
normetanephrine
Penicillium simplicissimum
-
-
0.0002
p-Cresol
Penicillium simplicissimum
-
pH 7.5, 25C, mutant enzyme D170E
0.26
propylphenol
Penicillium simplicissimum
-
pH 7.5, 25C, mutant enzyme D170E
-
0.004 - 5.4
Vanillyl alcohol
0.02
vanillylamine
Penicillium simplicissimum
-
pH 7.4, 25C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
102
Br-
-
pH 7.5, 25C
120
Cl-
-
pH 7.5, 25C
70
Thiocyanate
-
pH 7.5, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
-
flavinylation assay at
7 - 8
-
reaction with creosol
7.5
-
oxidase assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
-
pH 6.0: about 60% of maximal activity, pH 9.0: about 65% of maximal activity, reaction with creosol
7 - 12
-
about 30% of maximal activity at pH 7 and pH 12
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
flavinylation and oxidase assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
dual location: in peroxisomes and in cytosol
Manually annotated by BRENDA team
-
dual location: in peroxisomes and in cytosol
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
62790
-
monomeric apoVAO, mass spectrometry
125600
-
dimeric apoVAO, mass spectrometry
504700
-
wild-type enzyme, nanoelectrospray mass spectrometry
520000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 62915, calculation from nucleotide sequence
dimer
-
2 x 62786, apoVAO, mass spectrometry
monomer
-
1 x * 62786, apoVAO, mass spectrometry
octamer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
flavoprotein
-
covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the enzyme in the native state and in complexes with the four inhibitors p-cresol, isoeugenol, 2-nitro-p-cresol and heptenyl-phenol
-
crystals of mutant enzyme D170S are grown using hanging-drop vapor diffusion method
-
crystals of mutant enzyme D170S/T457E are grown by using the hanging-drop vapor diffusion method
-
hanging-drop vapor diffusion method, apoenzyme, ADP-complex and holoenzyme. Crystal structure of both the holo and apo form of H61T are highly similar to the structure of the wild-type enzyme
-
hanging-drop vapor diffusion method. Crystal structures of mutant enzymes I238T, F454Y, E502G and T505S are highly similar to that of wild-type enzyme
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
90 min, stable
60
-
45 min, 50% loss of activity
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete irreversible bleaching of the flavin spectrum
-
10666
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C or 4C, in 20% (NH4)2SO4, pH 7.0, stable for several weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in the vanillin-tolerant Gram-positive Amycolatopsis spec. HR167
-
mutant enzymes I238T, F454Y, E520G and T505S are overexpressed in Escherichia coli
-
overexpression of apoVAO in Escherichia coli strain BSV11, which is defective in riboflavin synthesis. Incubation of apoVAO with FAD results in full restoration of enzyme activity dependent on FAD concentration, displaying a hyperbolic relationship, overview
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D170A/T457E
-
produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol
D170S/T457E
-
produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol
E502G
-
the octamer/dimer ratio is 1:10. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols
F454Y
-
as for wild-type enzyme the octamer/dimer ratio of the mutant enzyme is 1.5:1. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols
H422A
-
mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Mutant enzyme is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD
H422C
-
mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD
H422T
-
mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD
I238T
-
the octamer/dimer ratio is 4:1. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols
T505S
-
as for wild-type enzyme the octamer/dimer ratio of the mutant enzyme is 1.5:1
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis