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Information on EC 1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase and Organism(s) Acinetobacter baumannii and UniProt Accession Q6Q272

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IUBMB Comments
The enzyme from Escherichia coli attacks a broad spectrum of phenolic compounds. The enzyme uses FADH2 as a substrate rather than a cofactor . FADH2 is provided by EC 1.5.1.36, flavin reductase (NADH) [5,6].
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Acinetobacter baumannii
UNIPROT: Q6Q272
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Word Map
The taxonomic range for the selected organisms is: Acinetobacter baumannii
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
hpabc, 4-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate hydroxylase, 4-hydroxyphenylacetate 3-monooxygenase, 4-hpa hydroxylase, 4-hydroxyphenylacetic acid 3-hydroxylase, 4hpa3h, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
HPA 3-hydroxylase
-
p-hydroxyphenylacetate 3-hydroxylase
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p-hydroxyphenylacetate hydroxylase
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4 HPA 3-hydroxyylase
-
-
-
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4-hydroxyphenylacetate 3-hydroxylase
4-hydroxyphenylacetic acid 3-hydroxylase
-
-
-
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HPA 3-hydroxylase
-
-
p-hydroxyphenylacetate 3-hydroxylase
p-hydroxyphenylacetate hydroxylase
p-hydroxyphenylacetic 3-hydroxylase
-
-
-
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two-component p-hydroxyphenylacetate hydroxylase
-
-
additional information
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
catalytic mechanism and structure-function relationship, overview
4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
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oxidation
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-
-
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reduction
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-
-
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hydroxylation
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-
-
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SYSTEMATIC NAME
IUBMB Comments
4-hydroxyphenylacetate,FAD:oxygen oxidoreductase (3-hydroxylating)
The enzyme from Escherichia coli attacks a broad spectrum of phenolic compounds. The enzyme uses FADH2 as a substrate rather than a cofactor [4]. FADH2 is provided by EC 1.5.1.36, flavin reductase (NADH) [5,6].
CAS REGISTRY NUMBER
COMMENTARY hide
37256-71-6
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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
3-hydroxyphenylacetate + FMNH2 + O2
3,4-hydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
hydroxylation rate constant is 16 per s and the product conversion ratio is 90%
-
?
4-aminophenylacetate + FMNH2 + O2
4-amino-3,5-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
?
4-hydroxyphenylacetate + FMNH + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
?
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
octopamine + FMNH + O2
norepinephrine + FMN + H2O
show the reaction diagram
no substrate of wild-type or mutant R263E, R263D, R263A, but substrate of mutant R263D/Y398D
-
-
?
tyramine + FMNH + O2
dopamine + FMN + H2O
show the reaction diagram
no substrate of wild-type or mutant R263E, but substrate of mutants R263D and R263A
-
-
?
3-hydroxyphenylacetate + NAD(P)H + O2
3,4-hydroxyphenylacetate + NAD(P)+ + H2O
show the reaction diagram
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98% of 4-hydroxyphenylacetate activity
-
-
?
4-hydroxyphenylacetate + FADH2 + O2
3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + FMNH2 + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
phenylacetic acid + ?
?
show the reaction diagram
-
97% of 4-hydroxyphenylacetate activity
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-
?
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
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxyphenylacetate + FADH2 + O2
3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + FMNH2 + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
FMN and FAD can substitute for each other, required
FMNH2
involved in reaction step 2 catalyzed by the monooxygenase enzyme component C2
FMNH2
riboflavin
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component C1
additional information
-
hydroxyphenylacetate forms a dead end complex with the (C2-C4a)-hydroxy-FMN intermediate inhibiting the bound flavin from returning to the oxidized form, FADH2 is equally active, the enzyme oxygenase component C2 has the unusual ability to use both common flavin cofactors in catalysis, kinetics, overview
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxyphenylacetate
inhibition of the dehydration step by 4-hydroxyphenylacetate at pH 9.0
FAD
-
inhibitory effect at more than 0.15 mM
FMN
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inhibitory effect at more than 0.15 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxyphenylacetate
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15 - 26
4-hydroxyphenylacetate
21 - 28
NADH
0.014 - 26
4-hydroxyphenylacetate
0.012 - 28
NADH
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
343 - 389
4-hydroxyphenylacetate
343 - 389
4-hydroxyphenylacetate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
200
purified recombinant C1 component
8.3
purified recombinant C2 component
200
purified recombinant C1 component
201
-
C1 component
8.3
purified recombinant C2 component
8.89
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C2 component
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
substrate 4-aminophenylacetate can only bind to the wild-type enzyme at pH 6.0, substrate can bind to mutant S146A at both pH 6.0 and 9.0
9
substrate can bind to mutant S146A at both pH 6.0 and 9.0
6.2 - 9.9
assay range for determination of reaction kinetics of C2-FMNH with oxygen at various pH values investigated by stopped-flow and rapid quenched-flow techniques, detailed overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
HPAH_ACIBA
422
0
46950
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
47000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
209000
-
component C2, gel filtration
32000
-
x * 32000, small component, SDS-PAGE
35000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
47000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
50000
-
homotetramer, 4 * 50000, large component, SDS-PAGE
73000
-
component C1, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
?
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
dimer
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x * 32000, small component, SDS-PAGE
tetramer
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homotetramer, 4 * 50000, large component, SDS-PAGE
additional information
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
models of the homotetrameric C2 component in complex with oxygen and presence or absence of 4-hydroxyphenylacetate. In random-acceleration molecular dynamics simulations, substrate 4-hydroxyphenylacetate can only reach the active center after the C4a-hydroperoxyflavin derivative of FMNH- is formed, requiring uptake of O2 at the active site before 4-hydroxyphenylacetate
microbatch method, using 20% (W/v) PEG 400, 0.1 M sodium acetate pH 4.6 as a precipitant
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H120D
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120E
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120K
catalyzes hydroxylation with efficiency comparable to that of the wild-type enzyme, the hydroxylation rate constant for H120K is 5.7 per s and the product conversion ratio is 75%, compared to values of 16 s-1 and 90% for the wild-type enzyme
H120N
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120Q
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120R
mutant is able to catalyze hydroxylation
H120Y
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H396A
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 7.1 compared to 9.8 for wild-type
H396N
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 9.3 compared to 9.8 for wild-type
H396V
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 7.3 compared to 9.8 for wild-type
R263A
mutation of oxygenase component, 72% hydroxylation efficiency of phydroxyphenylacetate, 7% hydroxylation of tyramine
R263D
mutation of oxygenase component, variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%) while maintaining 86% hydroxylation efficiency of phydroxyphenylacetate
R263E
mutation of oxygenase component, 73% hydroxylation efficiency of phydroxyphenylacetate, no hydroxylation of tyramine
S146A
S146C
product formation decreases from about 65% at pH 6.0 to 27% at pH 10.0
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme components C1 and C2 from Escherichia coli strain BL21(DE3), C1 4.0fold, C2 1.81fold
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, phenyl Sepharose column chromatography, and G-25 gel filtration
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protamine sulfate precipitation, ion-exchange, gel filtration
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recombinant enzyme components C1 and C2 from Escherichia coli strain BL21(DE3), C1 4.0fold, C2 1.81fold
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
genes c1-hpah and c2-hpah, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3), the recombinant enzyme is similarly active cmpared to the native one
genes c1-hpah and c2-hpah, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3), the recombinant enzyme is similarly active compared to the native one
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Chaiyen, P.; Suadee, C.; Wilairat, P.
A novel two-protein component flavoprotein hydroxylase. p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii
Eur. J. Biochem.
268
5550-5561
2001
Acinetobacter baumannii, Escherichia coli, Pseudomonas putida
Manually annotated by BRENDA team
Thotsaporn, K.; Sucharitakul, J.; Wongratana, J.; Suadee, C.; Chaiyen, P.
Cloning and expression of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii: evidence of the divergence of enzymes in the class of two-protein component aromatic hydroxylases
Biochim. Biophys. Acta
1680
60-66
2004
Acinetobacter baumannii (Q6Q271), Acinetobacter baumannii (Q6Q272), Acinetobacter baumannii
Manually annotated by BRENDA team
Sucharitakul, J.; Chaiyen, P.; Entsch, B.; Ballou, D.P.
The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis
Biochemistry
44
10434-10442
2005
Acinetobacter baumannii
Manually annotated by BRENDA team
Sucharitakul, J.; Chaiyen, P.; Entsch, B.; Ballou, D.P.
Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii
J. Biol. Chem.
281
17044-17053
2006
Acinetobacter baumannii
Manually annotated by BRENDA team
Sucharitakul, J.; Phongsak, T.; Entsch, B.; Svasti, J.; Chaiyen, P.; Ballou, D.P.
Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase
Biochemistry
46
8611-8623
2007
Acinetobacter baumannii
Manually annotated by BRENDA team
Alfieri, A.; Fersini, F.; Ruangchan, N.; Prongjit, M.; Chaiyen, P.; Mattevi, A.
Structure of the monooxygenase component of a two-component flavoprotein monooxygenase
Proc. Natl. Acad. Sci. USA
104
1177-1182
2007
Acinetobacter baumannii (Q6Q272), Acinetobacter baumannii
Manually annotated by BRENDA team
Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Phongsak, T.; Sucharitakul, J.; Chaiyen, P.; Tanaka, F.
Ultrafast solvation dynamics of flavin mononucleotide in the reductase component of p-hydroxyphenylacetate hydroxylase
J. Phys. Chem. B
113
8439-8442
2009
Acinetobacter baumannii
Manually annotated by BRENDA team
Ruangchan, N.; Tongsook, C.; Sucharitakul, J.; Chaiyen, P.
pH-Dependent studies reveal an efficient hydroxylation mechanism of the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase
J. Biol. Chem.
286
223-233
2011
Acinetobacter baumannii, Acinetobacter baumannii (Q6Q272)
Manually annotated by BRENDA team
Tongsook, C.; Sucharitakul, J.; Thotsaporn, K.; Chaiyen, P.
Interactions with the substrate phenolic group are essential for hydroxylation by the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase
J. Biol. Chem.
286
44491-44502
2011
Acinetobacter baumannii (Q6Q272)
Manually annotated by BRENDA team
Oonanant, W.; Sucharitakul, J.; Chaiyen, P.; Yuvaniyama, J.
Crystallization and preliminary X-ray analysis of the reductase component of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii
Acta Crystallogr. Sect. F
68
720-723
2012
Acinetobacter baumannii
Manually annotated by BRENDA team
Phongsak, T.; Sucharitakul, J.; Thotsaporn, K.; Oonanant, W.; Yuvaniyama, J.; Svasti, J.; Ballou, D.P.; Chaiyen, P.
The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain
J. Biol. Chem.
287
26213-26222
2012
Acinetobacter baumannii
Manually annotated by BRENDA team
Dhammaraj, T.; Pinthong, C.; Visitsatthawong, S.; Tongsook, C.; Surawatanawong, P.; Chaiyen, P.
A single-site mutation at Ser146 expands the reactivity of the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase
ACS Chem. Biol.
11
2889-2896
2016
Acinetobacter baumannii (Q6Q272), Acinetobacter baumannii
Manually annotated by BRENDA team
Chenprakhon, P.; Dhammaraj, T.; Chantiwas, R.; Chaiyen, P.
Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase
Arch. Biochem. Biophys.
620
1-11
2017
Acinetobacter baumannii (Q6Q272), Acinetobacter baumannii
Manually annotated by BRENDA team
Chenprakhon, P.; Trisrivirat, D.; Thotsaporn, K.; Sucharitakul, J.; Chaiyen, P.
Control of C4a-hydroperoxyflavin protonation in the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase
Biochemistry
53
4084-4086
2014
Acinetobacter baumannii (Q6Q272)
Manually annotated by BRENDA team
Pietra, F.
Unveiling the pathways of dioxygen through the C2 component of the environmentally relevant monooxygenase p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii A molecular dynamics investigation
Chem. Biodivers.
13
954-960
2016
Acinetobacter baumannii (Q6Q272), Acinetobacter baumannii
Manually annotated by BRENDA team
Visitsatthawong, S.; Chenprakhon, P.; Chaiyen, P.; Surawatanawong, P.
Mechanism of oxygen activation in a flavin-dependent monooxygenase A nearly barrierless formation of C4a-hydroperoxyflavin via proton-coupled electron transfer
J. Am. Chem. Soc.
137
9363-9374
2015
Acinetobacter baumannii (Q6Q272)
Manually annotated by BRENDA team