Information on EC 1.17.99.1 - 4-methylphenol dehydrogenase (hydroxylating)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.17.99.1
-
RECOMMENDED NAME
GeneOntology No.
4-methylphenol dehydrogenase (hydroxylating)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-methylphenol + 2 acceptor + H2O = 4-hydroxybenzaldehyde + 2 reduced acceptor
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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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
Toluene degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
4-methylphenol:acceptor oxidoreductase (methyl-hydroxylating)
A flavocytochrome c (FAD). Phenazine methosulfate can act as acceptor. A quinone methide is probably formed as intermediate. The first hydroxylation forms 4-hydroxybenzyl alcohol; a second hydroxylation converts this into 4-hydroxybenzaldehyde.
CAS REGISTRY NUMBER
COMMENTARY hide
66772-07-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain NCIB 8955
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-
Manually annotated by BRENDA team
strain NCIB 8955
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Manually annotated by BRENDA team
denitrifying bacterium
PC-07
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-
Manually annotated by BRENDA team
denitrifying bacterium PC-07
PC-07
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Manually annotated by BRENDA team
strain NCIB 9867
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Manually annotated by BRENDA team
strain NCIB 9867
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-
Manually annotated by BRENDA team
gene pchF
UniProt
Manually annotated by BRENDA team
strain NCIB 9866
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-
Manually annotated by BRENDA team
NCIMB 9869
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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using a gene-knockout it is shown that PCMH-encoding gene pchF is necessary for the catabolism of 2,4-xylenol
metabolism
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
(RS)-1-(4-hydroxyphenyl)ethanol + acceptor + H2O
?
show the reaction diagram
-
-
-
-
?
1,4-hydroxyquinone + acceptor + H2O
?
show the reaction diagram
-
-
-
-
?
2,4-xylenol + acceptor + H2O
3-methyl-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
2-bromo-4-methylphenol + acceptor + H2O
3-bromo-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
-
-
-
-
?
2-methoxy-4-methylphenol + acceptor + H2O
3-methoxy-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
-
-
-
-
?
3,4-dimethylphenol + acceptor + H2O
2-methyl-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
3-fluoro-4-methylphenol + acceptor + H2O
2-fluoro-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
-
-
-
-
?
4-cresol + 2 acceptor + H2O
4-hydroxybenzaldehyde + 2 reduced acceptor
show the reaction diagram
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PCMH catalyzes both the hydroxylation of 4-cresol to 4-hydroxybenzyl alcohol and the subsequent oxidation of the latter to 4-hydroxybenzaldehyde in the presence of artificial electron acceptors, the in vivo function of PCMH is to oxidize both 4-cresol and 4-hydroxybenzyl alcohol
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-
?
4-cresol + acceptor + H2O
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
4-cresol + acceptor + H2O
4-hydroxybenzyl alcohol + reduced acceptor
show the reaction diagram
-
the obligately anaerobic Geobacter metallireducens uses the 4-cresol degradation pathway of denitrifying bacteria, PCMH catalyzes both 4-cresol hydroxylation and 4-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, unusually it is located in the membrane fraction
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-
?
4-cresol + azurin + H2O
4-hydroxybenzaldehyde + reduced azurin
show the reaction diagram
4-cresol + Fe3+ + H2O
4-hydroxybenzaldehyde + Fe2+
show the reaction diagram
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PCMH catalyzes both the hydroxylation of 4-cresol to 4-hydroxybenzyl alcohol and the subsequent oxidation of the latter to 4-hydroxybenzaldehyde in the presence of artificial electron acceptors
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-
?
4-cresol + ferricenium + H2O
4-hydroxybenzyl alcohol + ferrocenium
show the reaction diagram
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PCMH catalyzes both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation and uses NAD+, NADP+, ferricenium, or phenazine methosulfate as electron acceptors
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-
?
4-cresol + NAD(P)+ + H2O
4-hydroxybenzyl alcohol + NAD(P)H
show the reaction diagram
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PCMH catalyzes both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation and uses NAD+, NADP+, ferricenium, or phenazine methosulfate as electron acceptors
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-
?
4-cresol + nitrate + H2O
4-hydroxybenzaldehyde + ?
show the reaction diagram
denitrifying bacterium
-
nitrate is the natural terminal electron acceptor
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-
4-cresol + phenazine methosulfate + H2O
4-hydroxybenzyl alcohol + reduced phenazine methosulfate
show the reaction diagram
4-ethylphenol + acceptor + H2O
1-(4'-hydroxyphenyl)ethanol + reduced acceptor
show the reaction diagram
4-hydroxybenzyl alcohol + acceptor
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
4-hydroxybenzyl alcohol + Fe3+ + H2O
4-hydroxybenzaldehyde + Fe2+
show the reaction diagram
-
PCMH catalyzes both the hydroxylation of 4-cresol to 4-hydroxybenzyl alcohol and the subsequent oxidation of the latter to 4-hydroxybenzaldehyde in the presence of artificial electron acceptors
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-
?
4-isopropylphenol + acceptor + H2O
?
show the reaction diagram
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-
-
-
?
4-methyl-1-naphthol + acceptor + H2O
?
show the reaction diagram
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-
-
-
?
4-methyl-3-nitrophenol + acceptor + H2O
2-nitro-4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
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-
-
-
?
4-methylcatechol + acceptor + H2O
3,4-dihydroxybenzaldehyde + reduced acceptor
show the reaction diagram
4-methylphenol + acceptor + H2O
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
4-n-propylphenol + acceptor + H2O
?
show the reaction diagram
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-
-
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?
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
4-cresol + 2 acceptor + H2O
4-hydroxybenzaldehyde + 2 reduced acceptor
show the reaction diagram
-
PCMH catalyzes both the hydroxylation of 4-cresol to 4-hydroxybenzyl alcohol and the subsequent oxidation of the latter to 4-hydroxybenzaldehyde in the presence of artificial electron acceptors, the in vivo function of PCMH is to oxidize both 4-cresol and 4-hydroxybenzyl alcohol
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-
?
4-cresol + acceptor + H2O
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
4-cresol + acceptor + H2O
4-hydroxybenzyl alcohol + reduced acceptor
show the reaction diagram
-
the obligately anaerobic Geobacter metallireducens uses the 4-cresol degradation pathway of denitrifying bacteria, PCMH catalyzes both 4-cresol hydroxylation and 4-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, unusually it is located in the membrane fraction
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-
?
4-cresol + azurin + H2O
4-hydroxybenzaldehyde + reduced azurin
show the reaction diagram
4-cresol + nitrate + H2O
4-hydroxybenzaldehyde + ?
show the reaction diagram
denitrifying bacterium
-
nitrate is the natural terminal electron acceptor
-
-
-
4-hydroxybenzyl alcohol + acceptor
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
-
PCMH catalyzes both the hydroxylation of 4-cresol to 4-hydroxybenzyl alcohol and the subsequent oxidation of the latter to 4-hydroxybenzaldehyde in the presence of artificial electron acceptors, the in vivo function of PCMH is to oxidize both 4-cresol and 4-hydroxybenzyl alcohol
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-
?
4-methylphenol + acceptor + H2O
4-hydroxybenzaldehyde + reduced acceptor
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxy-FAD
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strong binding but significantly reduced activity
8alpha-O-tyrosyl-FAD
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the covalently bound flavin is 8alpha-O-tyrosyl-FAD
cytochrome
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the enzyme complex shows a a unique alphaalpha'beta2 subunit composition, with beta representing a c-type cytochrome
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cytochrome c
heme
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wild type enzyme and recombinant enzyme contains covalently bound heme
menaquinone
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active in 4-hydroxybenzyl alcohol oxidation
phenazine methosulfate
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additional information
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only traces of activity with 6-Br-FAD, iso-FAD, 5-deaza-FAD, 6-amino-FAD, or 6-hydroxy-FAD as cofactor
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
copper
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contains one copper atom per molecule
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Hydroxybenzyl alcohol
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mixed-type inhibition of 4-cresol oxidation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02
2,4-dimethyl phenol
denitrifying bacterium
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0.0173
2,4-Xylenol
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0.27
3,4-dimethyl phenol
denitrifying bacterium
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0.072
3,4-xylenol
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0.0017 - 0.11
4-Cresol
0.36 - 2.17
4-Ethylphenol
0.0027 - 0.17
4-Hydroxybenzyl alcohol
0.26
4-Methylcatechol
denitrifying bacterium
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0.0264 - 6.76
phenazine methosulfate
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.4 - 121
4-Cresol
additional information
additional information
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.018 - 0.235
4-Hydroxybenzyl alcohol
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
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cell extract, substrate 4-hydroxybenzyl alcohol
0.025
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cell extract, substrate 4-cresol
0.432
denitrifying bacterium
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2.2
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purified soluble components including the active site of the enzyme membrane complex
6.78
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hydroxylase B
9.95
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hydroxylase A
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
assay at; assay at
9
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
low activity
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14000
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equilibrium sedimentation
99000
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hydroxylase B, equilibrium sedimentation
100000
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hydroxylase B, gel filtration
108000
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hydroxylase A, gel filtration
114000
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hydroxylase A, equilibrium sedimentation
115000
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sedimentation equilibrium ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals prepared by free interface diffusion method in 8% polyethylene glycol 8000
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native enzyme and alpha subunit, sitting drop vapor diffusion method
X-ray crystal structure at 2.5 resolution
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stable for a few days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
380fold purification of the soluble components including the active site of the enzyme membrane complex by membrane preparation, solubilization of the complex with lauryldimethylamine N-oxide, gel filtration, and hydrophobic interaction chromatography
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native and mutant enzymes from Escherichia coli
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partial
denitrifying bacterium
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recombinant enzyme
recombinant enzyme from Escherichia coli
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recombinant enzymes
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recombinant protein
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the 9200 Da c-type cytochrome subunit from Pseudomonas putida NCIMB 9869, overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC
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using Ni-NTA-chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli as a His-tagged fusion protein
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expressed in Escherichia coli BL21(DE3)
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expression in Escherichia coli JM109 and Pseudomonas putida RA4007
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expression of the flavoprotein subunit with non-covalently attached FAD in Escherichia coli
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overexpression of the flavoprotein in Escherichia coli
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the 9200 Da c-type cytochrome subunit from Pseudomonas putida NCIMB 9869 is overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, are unsuccessful, yielding relatively low levels of the protein
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the pchACXF operon encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, mapping of the transcriptional start sites of the pch operons, DNA and amino acid sequence determination and analysis, sequence comparisons, and phylogenetic analysis, gene pchF expression in Escherichia coli strain DH5alpha; the pchACXF operon encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, mapping of the transcriptional start sites of the pch operons, DNA and amino acid sequence determination and analysis, sequence comparisons, and phylogenetic analysis, gene pchF expression in Escherichia coli strain DH5alpha
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
different induction patterns of strains PC18 and PC24 are caused by differences in the PchR regulators of these strains; different induction patterns of strains PC18 and PC24 are caused by differences in the PchR regulators of these strains
PCMH of strain PC18 is induced during growth on phenol
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
apo-PchF[Y384F]
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the mutant Y384F of the flavoprotein subunit displays stoichiometric noncovalent FAD binding. The mutant flavoprotein subunit associates with the cytochrome subunit, although not as avidly as the wild-type flavoprotein subunit containing covalently bound FAD
R474K
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reduced rate of FAD binding, strongly reduced activity
Y384F
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strongly decreased activity
R474K
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reduced rate of FAD binding, strongly reduced activity
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis