Information on EC 1.14.14.20 - phenol 2-monooxygenase (FADH2)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.14.20
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RECOMMENDED NAME
GeneOntology No.
phenol 2-monooxygenase (FADH2)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
phenol + FADH2 + O2 = catechol + FAD + H2O
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
phenol,FADH2:oxygen oxidoreductase (2-hydroxylating)
The enzyme catalyses the ortho-hydroxylation of simple phenols into the corresponding catechols. It accepts 4-methylphenol, 4-chlorophenol, and 4-fluorophenol [1] as well as 4-nitrophenol, 3-nitrophenol, and resorcinol [3]. The enzyme is part of a two-component system that also includes an NADH-dependent flavin reductase. It is strictly dependent on FADH2 and does not accept FMNH2 [1,3]. cf. EC 1.14.13.7, phenol 2-monooxygenase (NADPH).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene pheA1
UniProt
Manually annotated by BRENDA team
gene pheA1
UniProt
Manually annotated by BRENDA team
gene pheA1(1); gene pheA1(1)
UniProt
Manually annotated by BRENDA team
gene pheA1(1); gene pheA1(1)
UniProt
Manually annotated by BRENDA team
strain P1
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-
Manually annotated by BRENDA team
strain P1
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
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
2,4-dichlorophenol + FADH2 + O2
2,4-dichlorocatechol + FAD + H2O
show the reaction diagram
2-aminophenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
2-chlorophenol + FADH2 + O2
2-chlorocatechol + FAD + H2O
show the reaction diagram
2-chlorophenol + O2 + NADPH
?
show the reaction diagram
2-methyl-phenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
3-aminophenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
3-chlorophenol + O2 + NADPH
?
show the reaction diagram
3-methylphenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
3-nitrophenol + NAD(P)H + H+ + O2
? + NAD(P)+ + H2O
show the reaction diagram
4-aminophenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
4-chlorophenol + FADH2 + O2
4-chlorocatechol + FAD + H2O
show the reaction diagram
4-chlorophenol + O2 + NADPH
?
show the reaction diagram
4-methyl-phenol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
4-methylphenol + FADH2 + O2
4-methylcatechol + FAD + H2O
show the reaction diagram
-
-
-
?
4-nitrophenol + NAD(P)H + H+ + O2
? + NAD(P)+ + H2O
show the reaction diagram
phenol + FADH2 + O2
catechol + FAD + H2O
show the reaction diagram
phenol + FMNH2 + O2
catechol + FMN + H2O
show the reaction diagram
phenol + NAD(P)H + H+ + O2
catechol + NAD(P)+ + H2O
show the reaction diagram
phenol + NADH + H+ + O2
catechol + NAD+ + H2O
show the reaction diagram
phenol + NADPH + O2
?
show the reaction diagram
-
first step of phenol degradation
-
-
?
phenol + NADPH + O2
catechol + NADP+ + H2O
show the reaction diagram
phenol + reduced ribloflavin + O2
catechol + riboflavin + H2O
show the reaction diagram
phloroglucinol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
pyrogallol + O2 + NADPH
?
show the reaction diagram
-
-
-
-
?
quinol + O2 + NADPH
1,2,4-trihydroxybenzene + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
resorcinol + NAD(P)H + H+ + O2
? + NAD(P)+ + H2O
show the reaction diagram
resorcinol + NADPH + O2
?
show the reaction diagram
-
-
-
-
?
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
2,4-dichlorophenol + FADH2 + O2
2,4-dichlorocatechol + FAD + H2O
show the reaction diagram
2-chlorophenol + FADH2 + O2
2-chlorocatechol + FAD + H2O
show the reaction diagram
4-chlorophenol + FADH2 + O2
4-chlorocatechol + FAD + H2O
show the reaction diagram
4-methylphenol + FADH2 + O2
4-methylcatechol + FAD + H2O
show the reaction diagram
A0A069AW73
-
-
-
?
phenol + FADH2 + O2
catechol + FAD + H2O
show the reaction diagram
phenol + NADPH + O2
?
show the reaction diagram
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first step of phenol degradation
-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FADH2
NADPH
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
1 mM activates by 27%; activates 13% at 1 mM
Mn2+
1 mM activates by 13%; activates 27% at 1 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxymercuribenzoate
complete inhibition at 0.02 mM
Ag+
0.02 mM completely inhibits; complete inhibition at 0.02 mM
Co2+
1 mM inhibits by 20%; 20% inhibition at 1 mM
Cu2+
0.02 mM completely inhibits; complete inhibition at 0.02 mM
FAD
with respect to the total phenol hydroxylase activity, concentrations higher than 0.01 mM inhibit the catalyzed reaction; with respect to the total phenol hydroxylase activity, concentrations higher than 0.01 mM inhibit the catalyzed reaction
Fe2+
1 mM inhibits by 49%; 49% inhibition at 1 mM
Fe3+
0.1 mM inhibits by 23%, 1 mM completely inhibits; complete inhibition at 1 mM
N-ethylmaleimide
0.1 mM inhibits by 38%, 1 mM completely inhibits; complete inhibition at 1 mM
Ni2+
0.1 mM inhibits by 79%, 1 mM completely inhibits; complete inhibition at 1 mM
p-hydroxymercuribenzoate
0.0005 mM inhibits by 53%,0.02 mM completely inhibits the enzymic activity
Zn2+
1 mM inhibits by 77%; 77% inhibition at 1 mM
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin reductase PheA2
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the two-protein system phenol hydroxylase consists of an oxygenase (PheA1) and a flavin reductase (PheA2). PheA1 shows almost no phenol hydroxylase activity when assayed at 50C and pH 7.0 in absence of PheA2. The PheA1-mediated conversion of phenol to catechol is strongly stimulated in presence of catalytic amounts of PheA2
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phenol hydroxylase PheA2
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PheA1-mediated conversion of phenol to catechol is strongly stimulated in the presence of catalytic amounts of PheA2
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0016 - 0.0134
FAD
0.0055 - 0.0691
FMN
0.271 - 0.606
NADPH
0.0055 - 0.0677
riboflavin
additional information
additional information
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kinetic analysis of PheA2, NADH-dependent activities of PheA2 with different electron acceptors, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
43
FAD
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
180
FMN
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
147
riboflavin
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
26900
FAD
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
20
32700
FMN
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
56
26700
riboflavin
Geobacillus thermoglucosidasius
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NADH-dependent reduction, 25C, pH 7.0, recombinant PheA2
351
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.004
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specific NADH:FAD reductase activity, pH 7.0 50C, in absence of phenol hydroxylase PheA2
0.32
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specific activity of PheA1 in presence of PheA2; specific NADH:FAD reductase activity, pH 7.0 50C, in presence of 1 mM phenol hydroxylase PheA2
41.7
purified recombinant enzyme PheA2, pH 6.8, 30C
802
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specific NADH:FAD reductase activity of purified recombinant PheA2, pH 7.0, 53C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.7
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NADH:FAD reductase activity of PheA2
6.8
; assay at; assay at
additional information
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the pH-optimum of the NADH:FAD reductase activity of PheA2 is pH 6.7
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
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activity range, profile overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 40
assay at; assay at
55
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NADH:FAD reductase activity of PheA2; phenol hydroxylase system
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 35
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10C: about 80% of activity maximum, 35C: about 50% of activity maximum
30 - 65
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activity range, profile overview
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.16
; sequence calculation
5.2
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isoelectric focusing; isoelectric focusing; PheA1 and PheA2, isoelectric focusing
5.36
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sequence calculation
5.75
; sequence calculation
6.29
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sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
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recombinant PheA2 protein, gel filtration
45000
gel filtration; recombinant PheA2, gel filtration
236000
gel filtration; recombinant PheA1, gel filtration
238000
non-denaturing-PAGE followed by staining with Coomassie Brilliant Blue
290000 - 310000
isozymes, gel filtration
additional information
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PheA1 has a MW of 120000 Da as determined by gel filtration. PheA2 has a MW of 35000 Da as determined by gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
oligomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant PheA2 containing bound FAD cofactor and purified reduced holo-PheA2 in complex with oxidized NAD, hanging drop vapor diffusion method, mixing of 0.002 ml of 16 mg/ml protein in 50 mM sodium phosphate buffer, pH 7.0, with 0.002 ml of reservoir solution containing 20-26% PEG 3350 and 0.4 M magnesium nitrate, and equilibration against 1 ml of reservoir solution, 20C, several days, crystal soakig in NADH or FAD solution, X-ray diffraction structure determination and analysis at 2.1-2.2 A resolution, modeling
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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PheA1, stable for 2 h; purified recombinant PheA1, completely stable for 2 h
65
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2 h, more than 65% of the NADH:FAD reductase activity of PheA2 is maintained; purified recombinant PheA2, 2 h, 65% NADH:FAD reductase activity remaining
70
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complete inactivation of Phea1 after 10 min; purified recombinant PheA1, complete inactivation within 10 min
85
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purified recombinant PheA2, 2 h, 15-20% NADH:FAD reductase activity remaining
88
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2 h, 15-20% of the NADH:FAD reductase activity of PheA2 is maintained
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, no significant loss of activity of the flavin reductase component PheA2 after 4 months
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-70C, purified recombinant PheA1 protein is not very stable when stored at -70C because it forms aggregates after thawing
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-70C, purified recombinant PheA2 protein is stable for 4 months when stored at -70C
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4C, purified recombinant PheA1 is stable when stored as a protein precipitate in 80% ammonium sulfate at 4C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
on a Ni2+ column, to electrophoretic homogeneity; on a Ni2+ column, to electrophoretic homogeneity; recombinant His6-tagged PheA1 from Escherichia coli strain M15; recombinant His6-tagged PheA2 from Escherichia coli strain M15 by nickel affinity chromatography
PheA1 and PheA2 expressed in recombinant Escherichia coli BL21 pLysS cells; recombinant PheA1 from Escherichia coli by anion exchange and hydroxyapatite chromatography, followed by gel filtration; recombinant PheA2 from Escherichia coli by anion exchange and hydroxyapatite chromatography, followed by gel filtration
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recombinant wild-type and selenomethionine-substituted PheA2 from Escherichia coli strain BL21(DE3)pLysS by protamine sulfate fractionation, hydrophobic interaction chromatography, dialysis, anion exchange chromatography, and preparative gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene pheA1, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli, cloning of the pheA1 promoter from Geobacillus thermoglucosidasius and use for enzyme expression in Escherichia coli strain Rosetta, tandem expression of genes pheA1 and pheA2; gene pheA2, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli strain Rosetta tandem expression of genes pheA1 and pheA2
gene pheA1, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression in Escherichia coli, coexpression with pheA2 is required for catalytic activity; gene pheA2, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression in Escherichia coli, coexpression with pheA1 is required for catalytic activity
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gene pheA1, quantitative real-time PCR-based expression and promoter activity analysis, recombinant expression in Corynebacterium glutamicum strain RES167, a heterologous host lacking the PheR regulator, coexpression of gene pheA1 with gene pheA2 and regulator gene pheR, subcloning in Escherichia coli strain DH5alpha
gene pheA1, recombinant expression in Escherichia coli, tandem expression with pheA2 does not result in PheA2 protein; gene pheA2, recombinant expression in Escherichia coli, tandem expression with pheA1 does not result in PheA2 protein; PheA1 and PheA2 are separately expressed in recombinant Escherichia coli BL21 pLysS cells
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gene pheA2, sequence comparisons, recombinant overexpression of wild-type and selenomethionine-substituted PheA2 in Escherichia coli strain BL21(DE3)pLysS
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genes pheA1(1-3), phylogenetic analysis, typical for genes of the peripheral degradation of aromatic compounds, pheA1(13) and pheA2(1-3) are not located within gene clusters for central ortho- and meta-cleavage pathway. All three gene sets are nearby to genes with function in (chloro)aromatic degradation
phenol hydroxylase is a two-component flavin-dependent monooxygenase, the two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome, DNA and amino acid sequence determination and analysis, recombinant expression of His6-tagged PheA1 in Escherichia coli strain M15 by nickel affinity chromatography; phenol hydroxylase is a two-component flavin-dependent monooxygenase, the two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome, DNA and amino acid sequence determination and analysis, recombinant expression of His6-tagged PheA2 in Escherichia coli strain M15; plasmid pQE30A2 expressing His6PheA2 protein transformed into Escherichia coli M15; plasmid pQE9A1 expressing His6PheA1 protein transformed into Escherichia coli M15
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
no repression by glucose or glycerol
the activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, encoded by genes pheA2-pheA1, is repressed substantially by succinate, quantitative RT-PCR reveals that expression of the phe genes is suppressed by succinate at the transcriptional level
the activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, is induced by phenol, quantitative RT-PCR reveals that expression of the phe genes is induced by phenol at the transcriptional level
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
environmental protection
synthesis