Information on EC 1.14.13.12 - benzoate 4-monooxygenase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

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EC NUMBER
COMMENTARY hide
1.14.13.12
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RECOMMENDED NAME
GeneOntology No.
benzoate 4-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
benzoate + NADPH + H+ + O2 = 4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
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-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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redox reaction
-
-
-
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reduction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Benzoate degradation
-
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Aminobenzoate degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
benzoate,NADPH:oxygen oxidoreductase (4-hydroxylating)
Requires Fe2+ and tetrahydropteridine.
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CAS REGISTRY NUMBER
COMMENTARY hide
39391-25-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene CYP53B1
-
-
Manually annotated by BRENDA team
white-rot fungus, a lignin-degrading basidiomycete, ATCC 34541
SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug target
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the enzyme is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes
metabolism
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the enzyme is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-chlorobenzoate + NADPH + O2
2-chloro-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
2-fluorobenzoate + NADPH + O2
2-fluoro-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
2-hydroxybenzoate + NADPH + O2
2,4-dihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
2-methylbenzoate + NADPH + O2
2-methyl-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
2-methylbenzoic acid + NADPH + O2
4-hydroxy-2-methylbenzoic acid + NADP+ + H2O
show the reaction diagram
-
-
-
?
3-chlorobenzoate + NADPH + O2
3-chloro-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
3-fluorobenzoate + NADPH + O2
3-fluoro-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
3-hydroxybenzoate + NADPH + O2
3,4-dihydroxybenzoate + NADP+ + H2O
show the reaction diagram
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-
-
?
3-hydroxybenzoic acid + NADPH + O2
3,4-dihydroxybenzoic acid + NADP+ + H2O
show the reaction diagram
reaction of 3-hydroxybenzoate 4-monooxygenase, EC 1.14.13.23
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-
?
3-methoxybenzoate + NADPH + O2
3-methoxy-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
?
3-methylbenzoate + NADPH + O2
3-methyl-4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
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-
-
?
3-methylbenzoic acid + NADPH + O2
4-hydroxy-3-methylbenzoic acid + NADP+ + H2O
show the reaction diagram
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-
-
?
4-chlorobenzoate + NADPH + O2
?
show the reaction diagram
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-
-
-
?
4-methylbenzoate + NADPH + O2
?
show the reaction diagram
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-
-
-
?
benzoate + NADPH + H+ + O2
2-hydro-1,2-dihydroxybenzoate + NADP+
show the reaction diagram
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cis-benzoate dihydrodiol is subsequently dehydrogenated to catechol which is cleaved further
-
?
benzoate + NADPH + H+ + O2
4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
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-
-
?
benzoate + NADPH + O2
4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
benzoic acid + NADPH + O2
4-hydroxybenzoic acid + NADP+ + H2O
show the reaction diagram
cinnamate + NADPH + O2
4-coumarate + NADP+ + H2O
show the reaction diagram
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very low turnover rate
-
?
nicotinate + NADPH + O2
?
show the reaction diagram
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-
-
-
?
picolinate + NADPH + O2
?
show the reaction diagram
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-
-
-
?
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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
benzoate + NADPH + H+ + O2
2-hydro-1,2-dihydroxybenzoate + NADP+
show the reaction diagram
A8I4E8, A8I4F1, A8I4F4
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cis-benzoate dihydrodiol is subsequently dehydrogenated to catechol which is cleaved further
-
?
benzoate + NADPH + O2
4-hydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
enzyme is involved in degradation of chlorinated benzoic acid derivatives e.g. 2-chlorobenzoate and 3-chlorobenzoate
-
?
benzoic acid + NADPH + O2
4-hydroxybenzoic acid + NADP+ + H2O
show the reaction diagram
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
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2fold stimulation of NADPH oxidation rate
NADH
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hydroxylation is not supported when NADH, NADPH, dithiothreitol, 2-mercaptoethanol and ascorbic acid are added alone to the reaction mixture, stimulation if added together with tetrahydropteridine
NADPH
tetrahydropteridine
additional information
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron
heme iron, the heme-binding domain FSFGPRSCVG at residues 421–430 is located in the C-terminal region
NaCl
10% is optimal NaCl concentration for Chromohalobacter sp. strain HS-2
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(E)-1-(4-methylpiperidin-1-yl)-3-phenylprop-2-en-1-one
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competitive inhibitor
2,2'-dipyridyl
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0.05 mM; 50% inhibition
2-phenylpyrimidine-5-carboxylic acid
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second best inhibitor among hit compounds, good behaviour in the spectral binding assay but weak antifungal activity
3-methyl-4-(1H-pyrrol-1-yl)benzoic acid
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best inhibitor among hit compounds, best in the spectral binding assay and has second best antifungal activity
4-Chlorobenzoate
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4-Methylbenzoate
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4-nitrobenzoate
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38% inhibition
8-hydroxyquinoline
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5 mM, complete inhibition
aminopterin
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0.5 mM, 10% inhibition
benzaldehyde
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benzoate methyl ester
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slight inhibition
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Benzyl acetate
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0.5 mM, 21% inhibition
benzyl alcohol
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0.5 mM, 10% inhibition
benzylformate
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0.5 mM, 10% inhibition
diethyldithiocarbamate
iodoacetamide
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5 mM, 43% inhibition
iodoacetate
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5 mM, 23% inhibition
KCN
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30 mM, 21% inhibition
m-Hydroxybenzoate
Mg2+
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0.001-0.01 mM, complete inhibition
Mn2+
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0.001-0.01 mM, complete inhibition
Mo2+
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0.001-0.01 mM, complete inhibition
N-ethylmaleimide
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o-phenanthroline
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0.05 mM, 70% inhibition
oxalate
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2 mM, 20% inhibition
p-chloromercuribenzoate
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p-hydroxymercuribenzoate
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0.052 mM, 39% inhibition
phenylalanine
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0.5 mM, 21% inhibition
Quinacrine
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0.5 mM, 47% inhibition
quinine-HCl
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0.5 mM, 42% inhibition
salicylate
SKF-525A
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cytochorme P450 inhibitor, 1 mM, 51% inhibition
trans-cinnamate
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0.5 mM, 55% inhibition
Zn2+
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0.001-0.01 mM, complete inhibition
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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hydroxylation is not supported when NADH, NADPH, dithiothreitol, 2-mercaptoethanol and ascorbic acid are added alone to the reaction mixture, stimulation when added together with tetrahydropteridine
ascorbic acid
dithiothreitol
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hydroxylation is not supported when NADH, NADPH, dithiothreitol, 2-mercaptoethanol and ascorbic acid are added alone to the reaction mixture, stimulation when added together with tetrahydropteridine
additional information
benzoic acid induces transcription of the gene encoding the enzyme and has a regulatory effect
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.31
2-Chlorobenzoate
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0.097
2-Fluorobenzoate
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0.28
2-Hydroxybenzoate
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1.6
2-Methylbenzoate
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0.127
3-chlorobenzoate
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0.086
3-Fluorobenzoate
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0.189
3-Methoxybenzoate
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0.673
3-methylbenzoate
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0.045
6,7-dimethyltetrahydropterin
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0.029 - 0.3
Benzoate
0.045
NADH
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0.019 - 0.16
NADPH
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.33
2-Chlorobenzoate
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4.33
2-Fluorobenzoate
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4.17
2-Hydroxybenzoate
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3.67
2-Methylbenzoate
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6.17
3-chlorobenzoate
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4.83
3-Fluorobenzoate
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4.5
3-hydroxybenzoate
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4
3-Methoxybenzoate
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6.17
3-methylbenzoate
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0.04 - 4.5
Benzoate
0.005
cinnamate
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reconstituted enzyme system consisting of benzoate 4-hydroxylase i.e. P450rm, cytochrome P450 reductase, NADPH and dilauroylphosphatidylcholine
4
nicotinate
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3.83
picolinate
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-
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00181
2-phenylpyrimidine-5-carboxylic acid
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pH 7.4, 22°C
0.0017
3-methyl-4-(1H-pyrrol-1-yl)benzoic acid
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pH 7.4, 22°C
0.75
4-nitrobenzoate
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-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.041
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activity in cells grown on benzoate
0.26
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activity in cells grown on benzoate, 2-chlorobenzoate or 3-chlorobenzoate
additional information
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recombinant enzyme expressed in Yarrowia lipolytica clones, overview
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 8
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approx. 35% of maximal activity at pH 4.5, approx. 15% of maximal activity at pH 8.0
5.5 - 8.2
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approx. 30% of maximal activity at pH 5.5, approx. 35% of maximal activity at pH 8.2
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 38
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rapid decrease of activity above 38°C and below 25°C
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
membrane associated
Manually annotated by BRENDA team
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
x * 60000, about, amino acid sequence calculation
120000
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gel filtration
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 60000, about, amino acid sequence calculation
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
molecular docking and 3D simulation of inhibition. Reaction mechanism assumes that the enzyme is membrane-bound and suggests that the hydrophobic ligands enter the active site through a channel, that is surrounded by numerous nonpolar residues including four prolines and is adjacent to the membrane-anchoring N-terminus
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7.5
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440256
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
benzoate stabilizes
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EDTA stabilizes
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freezing and thawing has no effect
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glutathione stabilizes
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quite stable in presence of benzoate
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0-4°C, enzyme concentration 0.050 mg/ml, pH 6.0-7.5, 15 days, no loss of activity
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0-4°C, pH 7.2-8.0, Tris-HCl buffer, 24 h, 50% loss of activity
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4°C, 24 h, 40% loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose, benzoate-agarose
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protamine sulfate, heat treatment, calcium phosphate gel, ammonium sulfate, DEAE-Sephadex, Sephadex G-150
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protamine sulfate, tricalcium phosphate gel, DEAE-cellulose, alumina C-gamma gel
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
coexpression of benzoate 4-hydroxylase and cytochrome P450 reductase in Aspergillus niger increases enzyme activity
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gene CYP1f, DNA and amino acid sequence determination and analysis, five introns and six exons, functional expression in Pichia pastoris
gene CYP53B1, functional overexpression in Yarrowia lipolytica strain UOFS Y-2366, multiple copies of CYP53B1 cDNA cloned under control of POX2 promoter, construction of several clones, feeding of stearic acid to the recombinant yeast leads to high accumulation of 4-hydroxybenzoate
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His-tagged benzoate 4-monooxygenase is expressed in Escherichia coli C43(DE3) cells
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PCR amplification, expression in Escherichia coli BL21 (DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
positively regulated at the transcriptional level by benzoic acid. Methyl benzoate and 4-aminobenzoate also act as inducers
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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the enzyme is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes
pharmacology
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the enzyme is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, a potential inhibitor-target system for antifungal drugs is developed
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LINKS TO OTHER DATABASES (specific for EC-Number 1.14.13.12)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)