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Information on EC 1.1.3.38 - vanillyl-alcohol oxidase and Organism(s) Penicillium simplicissimum and UniProt Accession P56216

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EC Tree
     1 Oxidoreductases
         1.1 Acting on the CH-OH group of donors
             1.1.3 With oxygen as acceptor
                1.1.3.38 vanillyl-alcohol oxidase
IUBMB Comments
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.
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This record set is specific for:
Penicillium simplicissimum
UNIPROT: P56216
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Word Map
The taxonomic range for the selected organisms is: Penicillium simplicissimum
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Reaction Schemes
Synonyms
vao, vanillyl-alcohol oxidase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4-allylphenol oxidase
-
-
-
-
4-hydroxy-2-methoxybenzyl alcohol oxidase
-
-
-
-
Aryl-alcohol oxidase
-
-
-
-
Oxidase, vanillyl alcohol
-
-
-
-
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
-
-
-
-
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
2-amino-p-cresol + O2
?
show the reaction diagram
-
-
-
?
2-methyl-p-cresol + O2
?
show the reaction diagram
-
-
-
?
4-(methoxymethyl)phenol + O2
?
show the reaction diagram
-
-
-
?
4-(methoxymethyl)phenol + O2 + H2O
4-hydroxybenzaldehyde + methanol + 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-cyclohexylphenol + O2
?
show the reaction diagram
-
-
-
?
4-cyclopentylphenol + O2
4-(1-cyclopenten-1-yl)phenol
show the reaction diagram
-
-
-
?
4-ethylphenol + O2 + H2O
(R)-1-(4'-hydroxyphenyl)ethanol + H2O2
show the reaction diagram
-
predominant product of wild-type enzyme and of mutant T457E
-
ir
4-ethylphenol + O2 + H2O
(S)-1-(4'-hydroxyphenyl)ethanol + H2O2
show the reaction diagram
-
predominant product of mutant D170S/T457E
-
ir
4-ethylphenol + O2 + H2O
1-(4'-hydroxyphenyl)ethanol + H2O2
show the reaction diagram
-
-
-
?
4-ethylphenol + O2 + H2O
? + H2O2
show the reaction diagram
-
-
-
?
4-n-propylphenol + O2
?
show the reaction diagram
-
-
-
?
chavicol + O2 + H2O
coumaryl alcohol + H2O2
show the reaction diagram
-
-
-
?
creosol + O2
?
show the reaction diagram
-
-
-
?
creosol + O2
vanillin + H2O
show the reaction diagram
-
-
-
?
eugenol + O2
?
show the reaction diagram
-
-
-
?
eugenol + O2 + H2O
coniferyl alcohol + H2O2
show the reaction diagram
-
-
-
?
p-cresol + O2
?
show the reaction diagram
-
-
-
?
vanillyl alcohol + O2
vanillin + H2O2
show the reaction diagram
(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-Methoxy-4-n-propylphenol + O2
1-(4'-Hydroxy-3'-methoxyphenyl)propanol
show the reaction diagram
3-(4'-Hydroxyphenyl)propanol + O2
?
show the reaction diagram
-
-
-
-
?
3-methoxy-4-hydroxybenzyl alcohol + O2
3-methoxy-4-hydroxybenzaldehyde + H2O2
show the reaction diagram
-
i.e. vanillyl alcohol
i.e. vanillin
-
?
4-(3'-methylcrotyl)phenol + O2
4-[(1E)-3-hydroxy-3-methylbut-1-en-1-yl]phenol + 4-[(1E)-3-methylbuta-1,3-dien-1-yl]phenol
show the reaction diagram
4-(methoxymethyl)phenol + O2
?
show the reaction diagram
-
-
-
-
?
4-(methoxymethyl)phenol + O2 + H2O
4-hydroxybenzaldehyde + methanol + H2O2
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
4-ethylphenol + O2 + H2O
(R)-1-(4'-hydroxyphenyl)ethanol + 4-vinylphenol + H2O2
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 + H2O
(R)-1-(4'-hydroxyphenyl)ethanol + H2O2
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-hydroxy-3-methoxybenzylamine + O2 + H2O
vanillin + H2O2 + NH3
show the reaction diagram
-
i.e. vanillylamine
-
-
?
4-hydroxy-3-methoxyphenylglycol + O2 + H2O
2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)ethan-1-one + H2O2
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-(2-hydroxypropan-2-yl)phenol + 4-isopropenylphenol
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-[(1E)-but-1-en-1-yl]phenol + 4-[(1Z)-but-1-en-1-yl]phenol
show the reaction diagram
-
-
93% cis-4-hydroxyphenylalkene + 7% trans-4-hydroxyphenylalkene
?
4-n-heptylphenol + O2
4-[(1E)-hept-1-en-1-yl]phenol + 4-[(1Z)-hept-1-en-1-yl]phenol
show the reaction diagram
-
-
50% cis-4-hydroxyphenylalkene + 50% trans-4-hydroxyphenylalkene
?
4-n-pentylphenol + O2
4-[(1E)-pent-1-en-1-yl]phenol + 4-[(1Z)-pent-1-en-1-yl]phenol + H2O2
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-(2-hydroxybutan-2-yl)phenol + 4-(but-1-en-2-yl)phenol + 4-[(2Z)-but-2-en-2-yl]phenol
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
?
show the reaction diagram
-
-
4% 4-hydroxyphenylalcohol + 94% 4-hydroxyphenylalkene + 2% 4-hydroxyphenylalkanone
?
5-indanol + O2
?
show the reaction diagram
-
-
16% 4-hydroxyphenylalcohol + 24% 4-hydroxyphenylalkene + 60% 4-hydroxyphenylalkanone
?
chavicol + O2 + H2O
coumaryl alcohol + H2O2
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 + H2O
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
-
-
-
-
?
propylphenol + O2
?
show the reaction diagram
-
-
-
-
r
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 SUBSTRATE
NATURAL PRODUCT
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
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
chavicol
substrate inhibition
isoeugenol
competitive inhibitor, isoeugenol is predominantly in its deprotonated form when bound to wild-type VAOA, but predominantly in its protonated form when bound to the variants Y108F or Y503F
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
0.029 - 0.254
2-amino-p-cresol
0.001 - 0.021
2-methyl-p-cresol
0.004 - 238
4-(Methoxymethyl)phenol
0.012
4-Ethylphenol
wild type enzyme, at pH 7.5 and 25°C
0.0065 - 0.074
chavicol
0.001 - 0.041
creosol
0.0002 - 0.019
eugenol
0.031
p-Cresol
pH 7.5, 25°C, wild-type enzyme
0.006 - 1.75
Vanillyl alcohol
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'-Hydroxyphenyl)-2-butanone
-
-
0.1
2-(4'-hydroxyphenyl)ethanol
-
-
0.006
2-Methoxy-4-n-propylphenol
-
-
0.065
4-(3'-methylcrotyl)phenol
-
-
0.113
4-(Methoxymethyl)phenol
-
pH 7.5, 25°C, mutant enzyme D170E
0.006
4-butylphenol
-
pH 7.5, 25°C, 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
-
-
0.042
4-n-heptylphenol
-
-
0.008
4-n-pentylphenol
-
-
0.01
4-n-Propylphenol
-
pH 7.5, 25°C, 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
-
-
0.05
creosol
-
pH 7.4, 25°C
0.002 - 0.0049
eugenol
1.6
metanephrine
-
-
2.9
norepinephrine
-
-
1.4
normetanephrine
-
-
0.043
phenazine methosulfate
-
-
0.29 - 0.73
Vanillyl alcohol
0.048
vanillylamine
-
pH 7.4, 25°C
additional information
additional information
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.001 - 0.099
2-amino-p-cresol
0.002 - 0.063
2-methyl-p-cresol
0.02 - 3.9
4-(Methoxymethyl)phenol
3.7
4-cyclopentylphenol
His-tagged recombinant enzyme, pH 7.5, 25°C
-
2.6
4-Ethylphenol
wild type enzyme, at pH 7.5 and 25°C
0.13 - 7.4
chavicol
0.003 - 0.17
creosol
0.1 - 39
eugenol
0.042 - 2.6
Vanillyl alcohol
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'-Hydroxyphenyl)-2-butanone
-
-
0.004
2-(4'-hydroxyphenyl)ethanol
-
-
3.2
2-Methoxy-4-n-propylphenol
-
-
4.8
2-methoxy-4-propylphenol
-
-
0.1
3-(4'-hydroxyphenol)propanol
-
-
1.4
4-(3'-methylcrotyl)phenol
-
-
5.3
4-(methoxymethyl)-phenol
-
-
0.002 - 0.07
4-(Methoxymethyl)phenol
0.0001 - 0.12
4-butylphenol
0.0001 - 2.7
4-Ethylphenol
1.3
4-Hydroxy-3-methoxybenzylamine
-
-
3.6
4-hydroxy-3-methoxyphenylglycol
-
-
0.13 - 1.3
4-isopropylphenol
0.005
4-Methylphenol
-
-
1.2
4-n-butylphenol
-
-
0.3
4-n-pentylphenol
-
-
0.0002 - 4.2
4-n-Propylphenol
3.9
4-Propylphenol
-
-
0.05 - 0.5
4-sec-butylphenol
0.7
5,6,7,8-tetrahydro-2-naphthol
-
-
0.5
5-indanol
-
-
0.07
creosol
-
pH 7.4, 25°C
0.01 - 13
eugenol
0.8
metanephrine
-
-
0.3
norepinephrine
-
-
0.7
normetanephrine
-
-
0.0002
p-Cresol
-
pH 7.5, 25°C, mutant enzyme D170E
0.26
propylphenol
-
pH 7.5, 25°C, mutant enzyme D170E
0.004 - 5.4
Vanillyl alcohol
0.02
vanillylamine
-
pH 7.4, 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
217
4-Ethylphenol
wild type enzyme, at pH 7.5 and 25°C
1.8 - 1138
chavicol
2821
eugenol
wild type enzyme, at pH 7.5 and 25°C
0.024 - 19
Vanillyl alcohol
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.45 - 7
chavicol
102
Br-
-
pH 7.5, 25°C
120
Cl-
-
pH 7.5, 25°C
70
thiocyanate
-
pH 7.5, 25°C
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
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
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
VAOX_PENSI
560
0
63035
Swiss-Prot
other Location (Reliability: 2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
114000
gel filtration, mutant lacking a loop at the dimer-dimer interface
125600
dimeric apoVAO, mass spectrometry
485000
gel filtration, wild-type
510000
gel filtration
61000
8 * 61000, SDS-PAGE
62786
1 x * 62786, apoVAO, mass spectrometry
62790
monomeric apoVAO, mass spectrometry
62915
x * 62915, calculation from nucleotide sequence
504700
-
wild-type enzyme, nanoelectrospray mass spectrometry
520000
-
gel filtration
62742
-
8 * 62742, mutant enzyme H61T, apo-H61T: 15% octamer and 85% dimer, H61T + FAD: 78% octamer + 22% dimer, nanoelectrospray mass spectrometry
63561
-
8 * 63561, wild-type enzyme, 86% of the enzyme axists as octamer and 14% as dimer, nanoelectrospray mass spectrometry
65000
-
8 * 65000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 62915, calculation from nucleotide sequence
dimer
homooctamer
monomer
1 x * 62786, apoVAO, mass spectrometry
octamer
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
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
tyrosine residues Tyr-108 and Tyr-503 are positioned to facilitate deprotonation of substrate's phenol groups
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C470L
mutant displays similar activity to the wild-type enzyme with the substrates vanillyl alcohol, chavicol aund eugenol, but no activity with linear 4-alkylphenols
E502G
the octamer/dimer ratio is 1:10. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols
F424G
mutant does not contain any flavin after purification
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
I238T
the octamer/dimer ratio is 4:1. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols
I468V
mutant displays similar activity to the wild-type enzyme with the substrates vanillyl alcohol, chavicol aund eugenol, but no activity with linear 4-alkylphenols
L316M
mutant displays substrate specificity profile similar to wild-type
T457Q
mutant shows about 3fold increased activity towards vanillyl alcohol, but decrease in activity with all other substrates
T459I
mutant displays substrate specificity profile similar to wild-type
T505S
as for wild-type enzyme the octamer/dimer ratio of the mutant enzyme is 1.5:1
Y108F
deprotonation of the substrate's phenol group is impaired
Y108F/Y503F
deprotonation of the substrate's phenol group is impaired
Y503F
deprotonation of the substrate's phenol group is impaired
D170A
D170A/T457E
-
produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol
D170E
D170N
D170S
D170S/T457E
-
produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol
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
additional information
exchange of a loop at the dimer-dimer interface in octameric vanillin oxidase that is not present in dimeric EUGO. A vanillin oxidase variant where the loop was deleted, loopless VAO, exclusively forms dimers. Introduction of the loop into EUGO is not sufficient to induce its octamerization. Neither variant displays major changes in its catalytic properties as compared to the wild-type enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52
mutant Y108F/Y503F, midpoint of unfolding
53
mutants Y108F and Y503F, midpoint of unfolding
54.6
melting temperature
55
wild-type, midpoint of unfolding
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
-20°C or 4°C, in 20% (NH4)2SO4, pH 7.0, stable for several weeks
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, phenyl Sepharose column chromatography, and hydroxyapatite column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 cells
expression in Escherichia coli
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
expressed in the vanillin-tolerant Gram-positive Amycolatopsis spec. HR167
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method
synthesis
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Van der Heuvel, R.H.H.; Fraaije, M.W.; Laane, C.; van Berkel, W.J.H.
Regio- and stereospecific conversion of 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase
J. Bacteriol.
180
5646-5651
1998
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; Veeger, C.; van Berkel, W.J.H.
Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols
Eur. J. Biochem.
234
271-277
1995
Penicillium simplicissimum
Manually annotated by BRENDA team
De Jong, E.; van Berkel, W.J.H.; van der Zwan, R.P.; de Bont, J.A.M.
Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD
Eur. J. Biochem.
208
651-657
1992
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; Sjollema, K.A.; Veenhuis, M.; van Berkel, W.J.H.
Subcellular localization of vanillyl-alcohol oxidase in Penicillium simplicissimum
FEBS Lett.
422
65-68
1998
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; Mattevi, A.; van Berkel, W.J.H.
Mercuration of vanillyl-alcohol oxidase from Penicillium simplicissimum generates inactive dimers
FEBS Lett.
402
33-35
1997
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; van Berkel, W.J.H.
Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a p-quinone methide intermediate
J. Biol. Chem.
272
18111-18116
1997
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; van der Heuvel, R.H.H.; Roelofs, J.C.A.A.; van Berkel, W.J.H.
Kinetic mechanism of vanillyl-alcohol oxidase with short-chain 4-alkylphenols
Eur. J. Biochem.
253
712-719
1998
Penicillium simplicissimum
Manually annotated by BRENDA team
Drijfhout, F.P.; Fraaije, M.W.; Jongejan, H.; van Berkel, W.J.H.; Franssen, M.C.R.
Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase
Biotechnol. Bioeng.
59
171-177
1998
Penicillium simplicissimum
Manually annotated by BRENDA team
Mattevi, A.; Fraaije, M.W.; Mozzarelli, A.; Olivi, L.; Coda, A.; van Berkel, W.J.H.
Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity
Structure
5
907-920
1997
Penicillium simplicissimum
Manually annotated by BRENDA team
Benen, J.A.E.; Sanchez-Torres, P.; Wagemaker, M.J.; Fraaije, M.W.; van Berkel, W.J.; Visser, J.
Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase
J. Biol. Chem.
273
7865-7872
1998
Penicillium simplicissimum (P56216), Penicillium simplicissimum, Penicillium simplicissimum CBS170.90 (P56216), Penicillium simplicissimum CBS170.90
Manually annotated by BRENDA team
Mattevi, A.; Fraaije, M.W.; Coda, A.; van Berkel, W.J.
Crystallization and preliminary X-ray analysis of the flavoenzyme vanilly-alcohol oxidase from Penicillium simplicissimum
Proteins
27
601-603
1997
Penicillium simplicissimum
Manually annotated by BRENDA team
van den Heuvel, R.H.H.; Laane, C.; van Berkel, W.J.H.
Exploring the biocatalytic potential of vanillyl-alcohol oxidase by site-directed mutagenesis
Adv. Synth. Catal.
343
515-520
2001
Penicillium simplicissimum
-
Manually annotated by BRENDA team
van den Heuvel, R.H.; Fraaije, M.W.; van Berkel, W.J.
Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols
FEBS Lett.
481
109-112
2000
Penicillium simplicissimum
Manually annotated by BRENDA team
van den Heuvel, R.H.; Partridge, J.; Laane, C.; Halling, P.J.; van Berkel, W.J.
Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering
FEBS Lett.
503
213-216
2001
Penicillium simplicissimum
Manually annotated by BRENDA team
van den Heuvel, R.H.; Fraaije, M.W.; Laane, C.; van Berkel, W.J.
Enzymatic synthesis of vanillin
J. Agric. Food Chem.
49
2954-2958
2001
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; van den Heuvel, R.H.; van Berkel, W.J.; Mattevi, A.
Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase
J. Biol. Chem.
274
35514-35520
1999
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
van den Heuvel, R.H.; Fraaije, M.W.; Mattevi, A.; van Berkel, W.J.
Asp-170 is crucial for the redox properties of vanillyl-alcohol oxidase
J. Biol. Chem.
275
14799-14808
2000
Penicillium simplicissimum
Manually annotated by BRENDA team
Fraaije, M.W.; van den Heuvel, R.H.; van Berkel, W.J.; Mattevi, A.
Structural analysis of flavinylation in vanillyl-alcohol oxidase
J. Biol. Chem.
275
38654-38658
2000
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Tahallah, N.; van den Heuvel, R.H.; van den Berg, W.A.; Maier, C.S.; van Berkel, W.J.; Heck, A.J.
Cofactor-dependent assembly of the flavoenzyme vanillyl-alcohol oxidase
J. Biol. Chem.
277
36425-36432
2002
Penicillium simplicissimum
Manually annotated by BRENDA team
van den Heuvel, R.H.; van den Berg, W.A.; Rovida, S.; van Berkel, W.J.
Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin
J. Biol. Chem.
279
33492-33500
2004
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
van den Heuvel, R.H.; Fraaije, M.W.; Ferrer, M.; Mattevi, A.; van Berkel, W.J.
Inversion of stereospecificity of vanillyl-alcohol oxidase
Proc. Natl. Acad. Sci. USA
97
9455-9460
2000
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Overhage, J.; Steinbuechel, A.; Priefert, H.
Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167
J. Biotechnol.
125
369-376
2006
Penicillium simplicissimum, Penicillium simplicissimum CBS 170.90
Manually annotated by BRENDA team
Jin, J.; Mazon, H.; van den Heuvel, R.H.; Heck, A.J.; Janssen, D.B.; Fraaije, M.W.
Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
FEBS J.
275
5191-5200
2008
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Ewing, T.A.; Gygli, G.; van Berkel, W.J.
A single loop is essential for the octamerization of vanillyl alcohol oxidase
FEBS J.
283
2546-2559
2016
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Ewing, T.A.; Gygli, G.; van Berkel, W.J.H.
A single loop is essential for the octamerization of vanillyl alcohol oxidase
FEBS J.
283
2546-2559
2016
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Ewing, T.A.; Nguyen, Q.T.; Allan, R.C.; Gygli, G.; Romero, E.; Binda, C.; Fraaije, M.W.; Mattevi, A.; van Berkel, W.J.H.
Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase
J. Biol. Chem.
292
14668-14679
2017
Penicillium simplicissimum (P56216), Penicillium simplicissimum
Manually annotated by BRENDA team
Ewing, T.A.; van Noord, A.; Paul, C.E.; van Berkel, W.J.H.
A xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases
Molecules
23
164
2018
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team
Gygli, G.; Lucas, M.F.; Guallar, V.; van Berkel, W.J.H.
The ins and outs of vanillyl alcohol oxidase Identification of ligand migration paths
PLoS Comput. Biol.
13
e1005787
2017
Penicillium simplicissimum (P56216)
Manually annotated by BRENDA team