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
1.1.3.38
-
RECOMMENDED NAME
GeneOntology No.
vanillyl-alcohol oxidase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Vanillyl alcohol + O2 = vanillin + H2O2
show the reaction diagram
hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the reaction with 4-allylphenols
-
Vanillyl alcohol + O2 = vanillin + H2O2
show the reaction diagram
the shape of the active-site cavity controls substrate specificity by providing a size exclusion mechanism. Inside the cavity the substrate aromatic ring is positioned at an angle of 18 to the flavin ring
-
Vanillyl alcohol + O2 = vanillin + H2O2
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4-allylphenol oxidase
-
-
-
-
4-hydroxy-2-methoxybenzyl alcohol oxidase
-
-
-
-
Aryl-alcohol oxidase
-
-
-
-
Oxidase, vanillyl alcohol
-
-
-
-
vanillyl-alcohol oxidase
-
-
vanillyl-alcohol oxidase
Penicillium simplicissimum CBS 170.90
-
-
-
VAO
-
-
-
-
VAO
Penicillium simplicissimum CBS 170.90
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
143929-24-2
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Penicillium simplicissimum CBS 170.90
CBS 170.90
-
-
Manually annotated by BRENDA team
Penicillium simplicissimum CBS170.90
CBS170.90
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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
-
-
94% R-enantiomer of 1-(4'-hydroxy-3'-methoxyphenyl)propanol
-
2-Methoxy-4-n-propylphenol + O2
1-(4'-Hydroxy-3'-methoxyphenyl)propanol
show the reaction diagram
-
-
90% 4-hydroxyphenylalcohol + 10% 4-hydroxyphenylalkene. Less than 1% cis-4-hydroxyphenylalkene + more than 99% trans-4-hydroxyphenylalkene
-
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-(3'-Methylcrotyl)phenol + O2
4-Hydroxyphenylalcohol + 4-hydroxyphenylalkene
show the reaction diagram
-
-
40% 4-hydroxyphenylalcohol + 60% 4-hydroxyphenylalkene. 100% trans-4-hydroxyphenylalkene
-
4-(Methoxymethyl)phenol + O2
4-Hydroxybenzaldehyde
show the reaction diagram
-
-
-
-
-
4-(Methoxymethyl)phenol + O2
4-Hydroxybenzaldehyde
show the reaction diagram
-
upon reduction, a binary complex is produced composed of the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme
-
-
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
?
show the reaction diagram
P56216
-
-
-
?
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
1-(4'-Hydroxyphenyl)ethanol + 4-vinylphenol + 4-hydroxyacetophenone
show the reaction diagram
-
-
76% 4-hydroxyphenolalcohol + 24% 4-hydroxyphenylalkene
-
4-Ethylphenol + O2
1-(4'-Hydroxyphenyl)ethanol + 4-vinylphenol + 4-hydroxyacetophenone
show the reaction diagram
-
-
94% R-enantiomer of 1-(4'-hydroxyphenyl)ethanol. 78% 1-(4'-hydroxyphenyl)ethanol + 18% 4-vinylphenol + 4% 4-hydroxyacetophenone
-
4-Ethylphenol + O2
1-(4'-Hydroxyphenyl)ethanol + 4-vinylphenol + 4-hydroxyacetophenone
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-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-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-Pentylphenol + O2
4-Hydroxyphenylalkene
show the reaction diagram
-
-
60% cis-4-hydroxyphenylalkene + 40% trans-4-hydroxyphenylalkene
-
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
1-(4'-Hydroxyphenyl)propanol + 4-propenylphenol + 1-(4'-hydroxyphenyl)propanone
show the reaction diagram
-
-
-
-
-
4-Propylphenol + O2
1-(4'-Hydroxyphenyl)propanol + 4-propenylphenol + 1-(4'-hydroxyphenyl)propanone
show the reaction diagram
-
-
94% R-enantiomer of 1-(4'-hydroxyphenyl)propanol. 78% 1-(4'-hydroxyphenyl)propanol + 18% 4-propenylphenol + 4% 1-(4'-hydroxyphenyl)propanone
-
4-Propylphenol + O2
1-(4'-Hydroxyphenyl)propanol + 4-propenylphenol + 1-(4'-hydroxyphenyl)propanone
show the reaction diagram
-
4-n-propylphenol
45% cis-4-hydroxyphenylalkene + 55% trans-4-hydroxyphenylalkene
-
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-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
coniferyl alcohol + H2O2
show the reaction diagram
-
-
-
-
eugenol + O2
coniferyl alcohol + H2O2
show the reaction diagram
-
-
-
-
?
eugenol + O2
?
show the reaction diagram
-
-
-
-
?
eugenol + O2
coniferyl alcohol + H2O
show the reaction diagram
Penicillium simplicissimum, Penicillium simplicissimum CBS 170.90
-
-
-
-
?
Norepinephrine + O2
?
show the reaction diagram
-
-
-
-
-
Normetanephrine + O2
?
show the reaction diagram
-
-
-
-
-
p-cresol + O2
?
show the reaction diagram
-
-
-
-
?
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
-
-
-
-
?
vanillyl alcohol + O2
?
show the reaction diagram
-
-
-
-
?
vanillyl alcohol + phenazine methosulfate
vanillin + reduced phenazine methosulfate
show the reaction diagram
-
-
-
-
vanillylamine + O2
?
show the reaction diagram
-
-
-
-
?
Metanephrine + O2
?
show the reaction diagram
-
-
-
-
-
additional information
?
-
-
enzyme also catalyzes the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols
-
-
-
additional information
?
-
-
short-chain 4-alkylphenols are predominantly hydroxylated to (R)-1-(4'-hydroxyphenyl) alcohols, whereas medium-chain 4-alkylphenols are dehydrogenated to the corresponding 1-(4'-hydroxyphenyl) alkenes
-
-
-
additional information
?
-
-
covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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 ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
8alpha-(N3-Histidyl)-FAD
-
the octamer contains 1 mol of covalently-bound flavin per mol of subunit
8alpha-(N3-Histidyl)-FAD
-
covalently bound prosthetic group
FAD
-
covalently bound in wild-type enzyme and mutant enzymes D170E and D170S, 50% of the FAD is covalently bound in mutant enzyme D170A and no FAD is covalently bound in mutant enzyme D170N
FAD
-
although mutations H422A, H422T or H422C, prevents covalent linkage of FAD, mutant enzymes contains tightly bound FAD. Covalent histidyl-FAD bond is not essential for FAD binding
FAD
-
covalently bound. Covalent flavinylation is an autocatalytical process in which His61 plays a crucial role by activating His422
FAD
-
a 8alpha-histidyl-C6-cysteinyl bound FAD, vanillyl-alcohol oxidase contains a covalently alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage, autocatalytic incorporation mechanism, overview. Incubation of recombinant apoVAO with FAD results in full restoration of enzyme activity dependent on FAD concentration, displaying a hyperbolic relationship with KFAD = 0.0023 mM, kactivation = 0.13/min. Formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate, overview
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 ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
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
-
-
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme T505S
0.043
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme E502G
0.05
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme I238T
0.064
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme F454Y
0.118
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme I238T
0.123
-
2-amino-p-cresol
-
pH 7.5, 25C, wild-type enzyme
0.167
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme F454Y
0.2
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme E502G
0.254
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme T505S
0.006
-
2-Methoxy-4-n-propylphenol
-
-
0.001
-
2-methyl-p-cresol
-
pH 10, 25C, mutant enzyme E502G
0.006
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme F454Y
0.007
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme E502G; pH 7.5, 25C, mutant enzyme T505S
0.017
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme I238T
0.021
-
2-methyl-p-cresol
-
pH 7.5, 25C, wild-type enzyme
0.065
-
4-(3'-Methylcrotyl)phenol
-
-
0.004
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme E502G
0.014
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme T505S
0.02
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme I238T
0.023
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme F454Y
0.033
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme T505S
0.034
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422A
0.037
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422C
0.04
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H61T
0.041
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422T
0.051
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme I238T
0.054
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme F454Y
0.058
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, wild-type enzyme
0.064
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme E502G
0.11
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, wild-type enzyme
0.113
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170E
55
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25Cm wild-type enzyme
65
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170A/T457E
168
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme T457E
238
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170S/T457E
0.006
-
4-butylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.007
-
4-Ethylphenol
-
-
0.009
-
4-Ethylphenol
-
-
0.048
-
4-Ethylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.24
-
4-Hydroxy-3-methoxybenzylamine
-
-
0.016
-
4-Isopropylphenol
-
-
0.026
-
4-Isopropylphenol
-
pH 7.5, 25C, mutant enzyme D170S
0.088
-
4-Isopropylphenol
-
pH 7.5, 25C, mutant enzyme D170E
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
-
4-Propylphenol
-
-
0.004
-
4-Propylphenol
-
-
0.062
-
4-sec-Butylphenol
-
pH 7.5, 25C, mutant enzyme D170S
0.072
-
4-sec-Butylphenol
-
-
0.075
-
4-sec-Butylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.094
-
5,6,7,8-Tetrahydro-2-naphthol
-
-
0.077
-
5-Indanol
-
-
0.0048
-
Chavicol
-
-
-
0.001
-
creosol
-
pH 10, 25C, mutant enzyme I238T; pH 10, 25C, mutant enzyme T505S
0.002
-
creosol
-
pH 10, 25C, mutant enzyme F454Y; pH 10, 25C, wild-type enzyme
0.013
-
creosol
-
pH 7.5, 25C, mutant enzyme F454Y
0.02
-
creosol
-
pH 7.5, 25C, wild-type enzyme
0.027
-
creosol
-
pH 7.5, 25C, mutant enzyme E502G
0.031
-
creosol
-
pH 7.5, 25C, mutant enzyme T505S
0.041
-
creosol
-
pH 7.5, 25C, mutant enzyme I238T
0.05
-
creosol
-
pH 7.4, 25C
0.0002
-
Eugenol
-
pH 10, 25C, mutant enzyme I238T
0.0008
-
Eugenol
-
pH 10, 25C, mutant enzyme F454Y; pH 7.5, 25C, mutant enzyme T505S
0.002
-
Eugenol
-
pH 7.5, 25C, mutant enzyme D170E; pH 7.5, 25C, mutant enzyme D170S
0.002
-
Eugenol
-
pH 10, 25C, mutant enzyme T505S; pH 7.5, 25C, wild-type enzyme
0.003
-
Eugenol
-
pH 7.5, 25C, mutant enzyme F454Y
0.005
-
Eugenol
-
pH 7.5, 25C, mutant enzyme E502G
0.008
-
Eugenol
-
pH 7.5, 25C, mutant enzyme I238T
0.019
-
Eugenol
-
pH 10, 25C, wild-type enzyme
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
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme T505S
0.007
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme F454Y; pH 7.5, 25C, mutant enzyme I238T
0.01
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme E502G; pH 10, 25C, mutant enzyme T505S
0.015
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme F454Y
0.022
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme I238T
0.051
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme E502G
0.075
-
Vanillyl alcohol
-
pH 7.5, 25C, wild-type enzyme
0.189
-
Vanillyl alcohol
-
pH 10, 25C, wild-type enzyme
0.29
-
Vanillyl alcohol
-
-
0.34
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme D170E
0.73
-
Vanillyl alcohol
-
-
0.048
-
vanillylamine
-
pH 7.4, 25C
1.6
-
metanephrine
-
-
additional information
-
additional information
-
-
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.7
-
(R)-1-(4'-hydroxyphenol)ethanol
-
-
3
-
(R,S)-1-(4'-Hydroxyphenyl)propanol
-
-
4.4
-
(S)-1-(4'-Hydroxyphenyl)ethanol
-
-
0.3
-
1-(4'-hydroxyphenol)-2-butanone
-
-
0.004
-
2-(4'-hydroxyphenyl)ethanol
-
-
0.001
-
2-amino-p-cresol
-
pH 10, 25C, wild-type enzyme
0.017
-
2-amino-p-cresol
-
pH 7.5, 25C, wild-type enzyme
0.046
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme T505S
0.047
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme F454Y
0.076
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme E502G
0.079
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme I238T
0.082
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme F454Y
0.086
-
2-amino-p-cresol
-
pH 10, 25C, mutant enzyme I238T
0.087
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme T505S
0.099
-
2-amino-p-cresol
-
pH 7.5, 25C, mutant enzyme E502G
3.2
-
2-Methoxy-4-n-propylphenol
-
-
4.8
-
2-methoxy-4-propylphenol
-
-
0.002
-
2-methyl-p-cresol
-
pH 10, 25C, wild-type enzyme
0.01
-
2-methyl-p-cresol
-
pH 10, 25C, mutant enzyme F454Y
0.019
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme E502G
0.02
-
2-methyl-p-cresol
-
pH 10, 25C, mutant enzyme I238T
0.023
-
2-methyl-p-cresol
-
pH 10, 25C, mutant enzyme T505S
0.025
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme T505S
0.028
-
2-methyl-p-cresol
-
pH 10, 25C, mutant enzyme E502G
0.031
-
2-methyl-p-cresol
-
pH 7.5, 25C, wild-type enzyme
0.032
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme F454Y
0.063
-
2-methyl-p-cresol
-
pH 7.5, 25C, mutant enzyme I238T
0.1
-
3-(4'-hydroxyphenol)propanol
-
-
1.4
-
4-(3'-Methylcrotyl)phenol
-
-
5.3
-
4-(methoxymethyl)-phenol
-
-
0.002
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170N
0.004
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170S
0.02
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170A/T457E
0.05
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170S/T457E
0.07
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme D170E
0.24
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H61T
0.27
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422A
0.28
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422T
0.32
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme H422C
1.1
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme E502G
1.2
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme E502G
1.3
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme T457E
2.1
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme F454Y; pH 7.5, 25C, mutant enzyme I238T
2.3
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, mutant enzyme T505S
3
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme I238T
3.1
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25C, wild-type enzyme
3.1
-
4-(Methoxymethyl)phenol
-
pH 7.5, 25Cm wild-type enzyme
3.4
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme F454Y
3.6
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, mutant enzyme T505S
3.9
-
4-(Methoxymethyl)phenol
-
pH 10, 25C, wild-type enzyme
0.0001
-
4-butylphenol
-
pH 7.5, 25C, mutant enzyme D170S
0.12
-
4-butylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.0001
-
4-Ethylphenol
-
pH 7.5, 25C, mutant enzyme D170S
0.17
-
4-Ethylphenol
-
pH 7.5, 25C, mutant enzyme D170E
1.3
-
4-Hydroxy-3-methoxybenzylamine
-
-
3.6
-
4-hydroxy-3-methoxyphenylglycol
-
-
0.13
-
4-Isopropylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.18
-
4-Isopropylphenol
-
pH 7.5, 25C, mutant enzyme D170S
1.3
-
4-Isopropylphenol
-
-
0.005
-
4-Methylphenol
-
-
1.2
-
4-n-Butylphenol
-
-
0.3
-
4-n-Pentylphenol
-
-
0.0002
-
4-n-Propylphenol
-
pH 7.5, 25C, mutant enzyme D170S
4.2
-
4-n-Propylphenol
-
-
3.9
-
4-Propylphenol
-
-
0.05
-
4-sec-Butylphenol
-
pH 7.5, 25C, mutant enzyme D170E
0.09
-
4-sec-Butylphenol
-
pH 7.5, 25C, mutant enzyme D170S
0.5
-
4-sec-Butylphenol
-
-
0.7
-
5,6,7,8-Tetrahydro-2-naphthol
-
-
6.5
-
Chavicol
-
-
-
0.003
-
creosol
-
pH 8, 25C, wild-type enzyme
0.005
-
creosol
-
pH 10, 25C, wild-type enzyme
0.01
-
creosol
-
pH 8, 25C, mutant enzyme T505S
0.013
-
creosol
-
pH 10, 25C, mutant enzyme F454Y; pH 8, 25C, wild-type enzyme
0.015
-
creosol
-
pH 10, 25C, mutant enzyme A429V; pH 8, 25C, mutant enzyme I237T/M437L
0.016
-
creosol
-
pH 8, 25C, mutant enzyme E502G
0.017
-
creosol
-
pH 10, 25C, mutant enzyme T505S; pH 8, 25C, mutant enzyme F454Y
0.018
-
creosol
-
pH 8, 25C, mutant enzyme A429V
0.019
-
creosol
-
pH 10, 25C, mutant enzyme F93Y/P189S/I238T
0.02
-
creosol
-
pH 10, 25C, mutant enzyme E502G; pH 10, 25C, mutant enzyme I238T; pH 7.5, 25C, wild-type enzyme
0.021
-
creosol
-
pH 10, 25C, mutant enzyme I237T/M437L
0.022
-
creosol
-
pH 8, 25C, mutant enzyme I238T
0.07
-
creosol
-
pH 7.4, 25C
0.07
-
creosol
-
pH 10, 25C, mutant enzyme I238T
0.09
-
creosol
-
pH 10, 25C, mutant enzyme E502G
0.1
-
creosol
-
pH 10, 25C, mutant enzyme F454Y; pH 10, 25C, mutant enzyme T505S; pH 7.5, 25C, mutant enzyme E502G
0.12
-
creosol
-
pH 7.5, 25C, mutant enzyme T505S
0.14
-
creosol
-
pH 7.5, 25C, mutant enzyme F454Y
0.17
-
creosol
-
pH 7.5, 25C, mutant enzyme I238T
2.7
-
ethylphenol
-
-
0.01
-
Eugenol
-
pH 7.5, 25C, mutant enzyme D170S
0.1
-
Eugenol
-
pH 10, 25C, mutant enzyme E502G
0.17
-
Eugenol
-
pH 7.5, 25C, mutant enzyme E502G
0.23
-
Eugenol
-
pH 10, 25C, mutant enzyme F454Y
0.34
-
Eugenol
-
pH 7.5, 25C, mutant enzyme D170E
0.5
-
Eugenol
-
pH 7.5, 25C, mutant enzyme F454Y
0.55
-
Eugenol
-
pH 10, 25C, mutant enzyme T505S
0.6
-
Eugenol
-
pH 10, 25C, mutant enzyme I238T
0.65
-
Eugenol
-
pH 7.5, 25C, mutant enzyme T505S
0.8
-
Eugenol
-
pH 7.5, 25C, mutant enzyme I238T
14
-
Eugenol
-
pH 7.5, 25C, wild-type enzyme
39
-
Eugenol
-
pH 10, 25C, wild-type enzyme
0.8
-
metanephrine
-
-
0.3
-
norepinephrine
-
-
0.7
-
normetanephrine
-
-
0.0002
-
p-Cresol
-
pH 7.5, 25C, mutant enzyme D170E
0.26
-
propylphenol
-
pH 7.5, 25C, mutant enzyme D170E
-
0.004
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme D170S
0.21
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme T505S
0.22
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme E502G
0.25
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme F454Y
0.3
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme E502G
0.39
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme I238T
0.67
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme T505S
0.71
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme F454Y
1.3
-
Vanillyl alcohol
-
pH 7.5, 25C, mutant enzyme D170E
1.4
-
Vanillyl alcohol
-
pH 10, 25C, mutant enzyme I238T
1.6
-
Vanillyl alcohol
-
pH 7.5, 25C, wild-type enzyme
2.4
-
Vanillyl alcohol
-
pH 10, 25C, wild-type enzyme
5.4
-
Vanillyl alcohol
-
-
0.02
-
vanillylamine
-
pH 7.4, 25C
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.8
-
-
flavinylation assay at
7
8
-
reaction with creosol
7.5
-
-
oxidase assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
flavinylation and oxidase assay at
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
62790
-
-
monomeric apoVAO, mass spectrometry
125600
-
-
dimeric apoVAO, mass spectrometry
504700
-
-
wild-type enzyme, nanoelectrospray mass spectrometry
520000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
P56216
x * 62915, calculation from nucleotide sequence
dimer
-
2 x 62786, apoVAO, mass spectrometry
octamer
-
8 * 65000, SDS-PAGE
octamer
-
8 * 62742, mutant enzyme H61T, apo-H61T: 15% octamer and 85% dimer, H61T + FAD: 78% octamer + 22% dimer, nanoelectrospray mass spectrometry; 8 * 63561, wild-type enzyme, 86% of the enzyme axists as octamer and 14% as dimer, nanoelectrospray mass spectrometry
monomer
-
1 x * 62786, apoVAO, mass spectrometry
additional information
-
FAD-free apoenzyme H61T mainly exists as a dimeric species of 126000 Da. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization
additional information
-
octamer/dimer ratio is 1.5:1 for wild-type enzyme and mutant enzymes F454Y and T505S. For I238T the octamer/dimer ratio is 4:1. For E502G the octamer/dimer ratio is 1:10
additional information
-
although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers, the enzyme is dimeric to about 80%
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
flavoprotein
-
covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
45
-
-
90 min, stable
60
-
-
45 min, 50% loss of activity
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
LITERATURE
-20C or 4C, in 20% (NH4)2SO4, pH 7.0, stable for several weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
D170A
-
only 50% of the FAD is covalently bound. With vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme
D170A
-
3100fold decrease in turnover-number for for 4-(methoxymethyl)phenol
D170A/T457E
-
produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol
D170E
-
with vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is 5-fold to 100fold less active than the wild-type enzyme
D170E
-
substrate preference is similar to wild-type enzyme, as the wild-type enzyme the mutant enzyme favors the production of alkenes
D170E
-
2fold increase in KM-value for for 4-(methoxymethyl)phenol, 2.1fold increase in Km-value for vanillyl alcohol, 2fold decrease in KM-value for eugenol as compared to wild-type enzyme.442fold decrease in turnover-number for for 4-(methoxymethyl)phenol, 2.5fold decrease in turnover-number for vanillyl alcohol, 51.5fold decrease in turnover-number for eugenol as compared to wild-type enzyme.Redox potential of mutant enzyme, + 6 mV, is decreased compared to wild-type enzyme, 55 mV
D170N
-
no FAD is covalently bound. With vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme
D170N
-
1550fold decrease in turnover-number for for 4-(methoxymethyl)phenol
D170S
-
with vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme
D170S
-
most active with branched-chain 4-alkylphenol, mutant enzyme favors the formation of alcohols
D170S
-
2fold decrease in KM-value for eugenol as compared to wild-type enzyme.1290fold decrease in turnover-number for for 4-(methoxymethyl)phenol, 825fold decrease in turnover-number for vanillyl alcohol, 1750fold decrease in turnover-number for eugenol as compared to wild-type enzyme. Redox potential of mutant enzyme, -91 mV, is decreased compared to wild-type enzyme, 55 mV
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
H61T
-
in the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The mutant enzyme is about 10fold less active with 4-(methoxymethyl)phenol than the wild-type enzyme. Crystal structure of both the holo and apo form of H61T are highly similar to the structure of the wild-type enzyme
H61T
-
FAD-free apoenzyme H61T mainly exists as a dimeric species of 126000 Da. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization
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 ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
synthesis of natural vanillin from capsaicin using a two-enzyme system of carboxylesterase from liver and vanillyl-alcohol oxidase
synthesis
-
bienzymatic route from capsaicin to vanillin for biocatalytic production of natural vanillin with carboxyesterase and vanillyl-alcohol oxidase