Information on EC 1.13.12.9 - phenylalanine 2-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.12.9
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RECOMMENDED NAME
GeneOntology No.
phenylalanine 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-phenylalanine + O2 = 2-phenylacetamide + CO2 + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Deamination
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-
-
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decarboxylation
oxidative decarboxylation
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-
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redox reaction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Phenylalanine metabolism
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SYSTEMATIC NAME
IUBMB Comments
L-phenylalanine:oxygen 2-oxidoreductase (decarboxylating)
The reaction shown above is about 80% of the reaction catalysed; the remaining 20% is:     L-phenylalanine + O2 + H2O = 3-phenylpyruvic acid + ammonia + H2O2 a reaction similar to that of EC 1.4.3.2, L-amino-acid oxidase.
CAS REGISTRY NUMBER
COMMENTARY hide
190396-37-3
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69403-11-8
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84012-76-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
beta-2-thienyl-DL-Ala + O2
? + CO2 + H2O
show the reaction diagram
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-
-
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?
beta-2-thienyl-DL-Ala + O2
? + NH3 + H2O2
show the reaction diagram
DL-m-Tyr + O2
? + CO2 + H2O
show the reaction diagram
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-
-
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?
DL-m-Tyr + O2
? + NH3 + H2O2
show the reaction diagram
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-
-
-
?
DL-o-Tyr + O2
? + CO2 + H2O
show the reaction diagram
DL-o-Tyr + O2
? + NH3 + H2O2
show the reaction diagram
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-
-
-
?
L-Ala + O2
? + CO2 + H2O
show the reaction diagram
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9% of the activity with L-Phe
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?
L-Ile + O2
? + CO2 + H2O
show the reaction diagram
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8% of the activity with L-Phe
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?
L-Met + O2
2-oxo-methiolbutyrate + NH3 + H2O2
show the reaction diagram
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-
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?
L-Met + O2
? + CO2 + H2O
show the reaction diagram
L-norleucine + O2
? + NH3 + H2O2
show the reaction diagram
-
-
-
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?
L-Phe + O2
2-phenylacetamide + CO2 + H2O
show the reaction diagram
L-Phe + O2
beta-phenylpyruvate + NH3 + H2O2
show the reaction diagram
L-phenylalanine + O2
2-phenylacetamide + CO2 + H2O
show the reaction diagram
L-Trp + O2
? + CO2 + H2O
show the reaction diagram
L-Tyr + O2
? + CO2 + H2O
show the reaction diagram
L-Tyr + O2
? + NH3 + H2O2
show the reaction diagram
p-fluoro-DL-Phe + O2
? + CO2 + H2O
show the reaction diagram
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-
-
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?
p-fluoro-DL-Phe + O2
? + NH3 + H2O2
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
L-Phe + O2
2-phenylacetamide + CO2 + H2O
show the reaction diagram
L-phenylalanine + O2
2-phenylacetamide + CO2 + H2O
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin adenine dinucleotide
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FAD
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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slight
2,2'-dipyridyl
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slight
2-amino benzoate
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3-Phenylpropionic acid
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competitive towards L-Phe
4-Phenylbutyric acid
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competitive towards L-Phe
5-phenylpentanoate
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8-hydroxyquinoline
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slight
CuSO4
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preferentially inhibits oxidase activity versus oxygenase activity
FeSO4
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preferentially inhibits oxidase activity versus oxygenase activity
phenylacetic acid
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inhibits both oxidation and oxygenation of L-Phe
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Pronase
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Trypsin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.052
beta-2-thienyl-DL-Ala
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calculation from oxygen consumption due to oxidation and/or oxygenation
0.071
beta-2-Thienylalanine
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-
0.048
DL-m-Tyr
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-
0.189
DL-o-Tyr
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calculation from oxygen consumption due to oxidation and/or oxygenation
0.502 - 2.2
L-Met
0.83
L-norleucine
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calculation from oxygen consumption due to oxidation and/or oxygenation
0.0107 - 0.1
L-Phe
0.0159 - 3.4
L-phenylalanine
0.275 - 4
L-Tyr
1.82 - 2.04
O2
0.012
p-fluoro-DL-Phe
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calculation from oxygen consumption due to oxidation and/or oxygenation
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
103 - 115
L-phenylalanine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.87
2-amino benzoate
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20 mM Tris-HCl, pH 8.0, 25°C
0.017
3-phenylpropanoate
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0.056
4-phenyl-n-butyrate
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0.93
5-phenylpentanoate
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7.1
D-Phe
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0.098
phenylacetic acid
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9.5
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formation of alpha-phenylacetamide
11
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formation of beta-phenylpyruvate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.8 - 11.8
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50% of maximal activity at pH 9.8 and 11.8, formation of beta-phenylpyruvate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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formation of alpha-phenylacetamide
65
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formation of beta-phenylpyruvate
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 70
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20°C: about 65% of maximal activity, 70°C: about 50% of maximal activity, formation of alpha-phenylacetamide
40 - 85
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40°C: about 50% of maximal activity 40°C and 85°C, formation of beta-phenylpyruvate
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
68000
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2 * 68000, SDS-PAGE
140000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
complexed with L-Phe and L-Met, vapor diffusion method, using 0.1 M HEPES (pH 7.5) and 1.0 M ammonium sulfate; crystal structure of PAOpt individually complexed with L-Phe and L-Met is constructed at constructed at 1.0-1.25 A resolution. The benzene ring of L-Phe is packed in six hydrophobic amino acid side chains versus the two hydrophobic side chains of L-amino acid oxidase. The distance between the substrate Calpha atom and water is shorter in the PAOpt-L-Met complex than in the PAOpt-L-Phe complex
crystals of noncatalytic proenzyme, activated enzyme, and enzyme o-amino benzoate complex, purified protein in 10 mM Tris-HCl (pH 8.0), concentrated, mixed with 0.1 M HEPES (pH 7.5) and 1.7 M ammonium sulfate (for the proenzyme) or 1.0 M ammonium sulfate (for the active enzyme), 30% glycerol in buffer as cryoprotectant, the enzyme amino benzoate complex is prepared by a soaking method with the cryoprotectant
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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10 min, no loss of activity
80
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10 min, 46% loss of activity
85
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10 min, 90% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 1 mM or 10 mM potassium phosphate buffer, pH 7.0, stable for at least 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell disruption by sonication, centrifugation, supernatant applied to HisTrap HP column equilibrated with 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl, elution with linear gradient of imidazole from 0-0.5 M, ammonium sulfate added to eluted fractions, centrifugation of precipitate, solution in 20 mM Tris-HCl (pH 8.0) containing 300 mM ammonium sulfate, several chromatographic steps using HiTrap Phenyl FF, Resource Q, and HiLoad 16/60 Superdex 200 prep grade, activated enzyme purified by HiTrap Q HP and gel filtration
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recombinant enzyme using His-tag
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) as His-tag fusion protein
expression of the proenzyme in Escherichia coli, site-directed mutation introduced on the wild-type proenzyme gene in the plasmid pPAO+15, expression of proenzyme mutants in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K478A
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catalytic activity 1/200 of the wild-type enzyme
M142A
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kcat is 1/10 of the wild-type enzyme, Km is elevated, affinity for oxygen seems decreased
R143A
inactive; mutant enzyme shows no activity
R143K
mutant enzyme shows 400fold lower activity compared to wild-type. Wild-type PAOpt produces higher amounts of phenylacetamide (PAM) than phenylpyruvate (PPV) as expected, but PAM is not produced by the R143K and Y536F mutants; the mutant has about 400fold lower activity than the wild type enzyme
Y536A
inactive; mutant enzyme shows no activity
Y536F
mutant enzyme shows 17fold lower activity compared to wild-type. Wild-type PAOpt produces higher amounts of phenylacetamide (PAM) than phenylpyruvate (PPV) as expected, but PAM is not produced by the R143K and Y536F mutants; the mutant has about 17fold lower activity than the wild type enzyme
K478A
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catalytic activity 1/200 of the wild-type enzyme
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M142A
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kcat is 1/10 of the wild-type enzyme, Km is elevated, affinity for oxygen seems decreased
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R143A
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inactive; mutant enzyme shows no activity
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R143K
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mutant enzyme shows 400fold lower activity compared to wild-type. Wild-type PAOpt produces higher amounts of phenylacetamide (PAM) than phenylpyruvate (PPV) as expected, but PAM is not produced by the R143K and Y536F mutants; the mutant has about 400fold lower activity than the wild type enzyme
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Y536F
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mutant enzyme shows 17fold lower activity compared to wild-type. Wild-type PAOpt produces higher amounts of phenylacetamide (PAM) than phenylpyruvate (PPV) as expected, but PAM is not produced by the R143K and Y536F mutants
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis