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Information on EC 1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase and Organism(s) Thermus thermophilus and UniProt Accession Q5SJP8
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1.14.14.9 4-hydroxyphenylacetate 3-monooxygenaseIUBMB 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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Word Map
- 1.14.14.9
- flavin
- 3,4-dihydroxyphenylacetate
- baumannii
-
flavin-dependent
- tyrosol
- hydroxytyrosol
- fmnh
-
two-protein
- piceatannol
- synthesis
The taxonomic range for the selected organisms is: Thermus thermophilus
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
hpabc, 4-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate hydroxylase, 4-hpa hydroxylase, 4-hydroxyphenylacetate 3-monooxygenase, 4-hydroxyphenylacetic acid 3-hydroxylase, 4hpa3h, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
4 HPA 3-hydroxyylase
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4-hydroxyphenylacetate 3-hydroxylase
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4-hydroxyphenylacetic acid 3-hydroxylase
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p-hydroxyphenylacetate 3-hydroxylase
p-hydroxyphenylacetate hydroxylase
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p-hydroxyphenylacetic 3-hydroxylase
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additional information
4-hydroxyphenylacetate 3-monooxygenase is a member of the two-component non-heme flavin-diffusible monooxygenase family
p-hydroxyphenylacetate 3-hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O
structural basis of the catalytic mechanism of HpaB
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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oxidation
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reduction
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hydroxylation
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PATHWAY SOURCE
PATHWAYS
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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
37256-71-6
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
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?
4-hydroxyphenylacetate + NADPH + H+ + O2
3,4-dihydroxyphenylacetate + NADP+ + H2O
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?
4-hydroxyphenylacetate + FMNH + O2
3,4-dihydroxyphenylacetate + FMN + H2O
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
4-hydroxyphenylacetate, FAD, NADH, and O2 binding structures, overview, the binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site involving Ser197 and Thr198. The latter loop further changes its conformation upon binding of 4-hydroxyphenylacetate and obstructs the active site from the bulk solvent. Arg100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4a-hydroperoxyflavin intermediate
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
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FAD and NAD+ are bound in the groove in the extended and folded conformation, respectively, structure, overview
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additional information
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the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
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?
additional information
?
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the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
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?
additional information
?
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the hydroxylation of 4-hydroxyphenylacetate by this oxygenase is performed in two stages. Firstly, the reductase component of the enzyme, the small 16.1 kDa protein HpaC, reduces flavin with the use of NAD(P)H. Subsequently, reduced flavin is transported to the oxygenase component HpaB of 4HPA 3-monooxygenase by free diffusion where, together with the oxygen molecule, it is used for the oxygenation of substrates. HpaB determines the substrate specificity
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?
additional information
?
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the hydroxylation of 4-hydroxyphenylacetate by this oxygenase is performed in two stages. Firstly, the reductase component of the enzyme, the small 16.1 kDa protein HpaC, reduces flavin with the use of NAD(P)H. Subsequently, reduced flavin is transported to the oxygenase component HpaB of 4HPA 3-monooxygenase by free diffusion where, together with the oxygen molecule, it is used for the oxygenation of substrates. HpaB determines the substrate specificity
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
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?
4-hydroxyphenylacetate + NADPH + H+ + O2
3,4-dihydroxyphenylacetate + NADP+ + H2O
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?
4-hydroxyphenylacetate + FMNH + O2
3,4-dihydroxyphenylacetate + FMN + H2O
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
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?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
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?
additional information
?
-
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
-
-
?
additional information
?
-
-
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
binding structure, binding causes conformational changes, overview
FAD
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FAD is not tightly bound to HpaC, the flavin reductase component of the enzyme, and is bound in the groove in the extended and folded conformation, binding site structure, overview
NAD(P)H
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product NAD+ is bound in the groove in the extended and folded conformation, binding site structure, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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the enzyme consists of two components, an oxygenase HpaB and a reductase HpaC, structure analysis, overview
additional information
the enzyme consists of two components, an oxygenase HpaB and a reductase HpaC, structure analysis, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, to 2.0, 1.66 and 1.66 A resolution, respectively. Binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site Ser197 and Thr198. The latter loop further changes its conformation upon binding of 4-hydroxyphenylacetate and obstructs the active site from the bulk solvent. Arg100 is located adjacent to the putative oxygen-binding site
sitting drop vapour diffusion method, 10 mg/ml protein in 0.01 M Tris-HCl, pH 7.6, equilibration against 0.5 ml of reservoir solution, different mixtures, overview, 20°C, well shaped single crystals, X-ray diffraction structure determination and analysis at 1.82 A resolution
flavin reductase component HpaC of 4-hydroxyphenylacetate 3-monooxygenase in a ternary complex with FAD and NAD+, mixing of 0.002-0.005 ml of a solution containing 25 mg/mL HpaCTt-FAD, 5 mM NADH, and 1 mM dithiothreitol with an equal volume of a reservoir solution containing 20% w/v PEG 1000, 10% w/v PEG 8000, and 10% v/v glycerol, and then equilibrating the solution against the reservoir solution, 2 days, X-ray diffraction structure determination and analysis at 1.65-3.3 A resolution
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HpaB in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, mixing of 0.004 ml of protein solution containing 5 mg/ml protein and 1 mM DTT, with an equal volume of reservoir solution containing 1.5 M ammonium sulfate, 0.1 M Tris-HCl, pH 8.5, and 25% v/v glycerol, crystals appear within a few min and grow during 1-4 days to final size, complex crystal formation by soaking of crystals in 5 mM ligand containing solutions, X-ray diffraction structure determination and analysis at 1.66-2.07 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S171A
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the mutant shows reduced activity compared to the wild type enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from membranes by solubilization with 0.1% Triton X-100, anion exchange chromatography, dialysis, exchange of detergent to dodecyl-beta-D-maltoside, another ion exchange chromatography, and gel filtration, further purification of HpaB by repetitive crystallization, method overview
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kim, S.H.; Hisano, T.; Takeda, K.; Iwasaki, W.; Ebihara, A.; Miki, K.
Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8
J. Biol. Chem.
282
33107-33117
2007
Thermus thermophilus, Thermus thermophilus (Q5SJP8), Thermus thermophilus HB8 / ATCC 27634 / DSM 579, Thermus thermophilus HB8 / ATCC 27634 / DSM 579 (Q5SJP8)
Kim, S.H.; Hisano, T.; Iwasaki, W.; Ebihara, A.; Miki, K.
Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity
Proteins
70
718-730
2008
Thermus thermophilus, Thermus thermophilus HB8 / ATCC 27634 / DSM 579
Soulimane, T.; OKane, S.R.; Kolaj, O.
Isolation and purification of Thermus thermophilus HpaB by a crystallization approach
Acta Crystallogr. Sect. F
66
352-356
2010
Thermus thermophilus (Q5SJP8), Thermus thermophilus, Thermus thermophilus HB8 / ATCC 27634 / DSM 579 (Q5SJP8)
Thotsaporn, K.; Chenprakhon, P.; Sucharitakul, J.; Mattevi, A.; Chaiyen, P.
Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms
J. Biol. Chem.
286
28170-28180
2011
Thermus thermophilus, Thermus thermophilus HB8 / ATCC 27634 / DSM 579
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