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Information on EC 1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase and Organism(s) Pseudomonas fluorescens and UniProt Accession P00438
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1.14.13.2 4-hydroxybenzoate 3-monooxygenaseIUBMB Comments
A flavoprotein (FAD). Most enzymes from Pseudomonas are highly specific for NADPH (cf. EC 1.14.13.33 4-hydroxybenzoate 3-monooxygenase [NAD(P)H]).
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Word Map
- 1.14.13.2
- flavin
- fad
- fluorescens
- flavoproteins
- isoalloxazine
- protocatechuate
-
3,4-dioxygenase
-
massey
- 2,4-dihydroxybenzoate
-
ballou
-
c4a-hydroperoxide
- 3-hydroxybenzoate
-
gatti
- degradation
- analysis
The taxonomic range for the selected organisms is: Pseudomonas fluorescens
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
p-hydroxybenzoate hydroxylase, para-hydroxybenzoate hydroxylase, 4-hydroxybenzoate hydroxylase, 4-hydroxybenzoate 3-monooxygenase, 4-hydroxybenzoate 3-hydroxylase, m-hydroxybenzoate hydroxylase, 4hba 3-hydroxylase, 4-hba 3-monooxygenase, 4-hba 3-hydroxylase, phbad, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
4-HBA-3-hydroxylase
-
-
-
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4-hydroxybenzoate 3-hydroxylase
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-
-
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4-hydroxybenzoate 3-monooxygenase
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-
-
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4-hydroxybenzoate monooxygenase
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-
-
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4-hydroxybenzoic hydroxylase
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-
-
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oxygenase, 4-hydroxybenzoate 3-mono-
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-
-
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p-hydroxybenzoate hydroxylase
p-hydroxybenzoate-3-hydroxylase
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-
-
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p-hydroxybenzoic acid hydrolase
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-
-
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p-hydroxybenzoic acid hydroxylase
-
-
-
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p-hydroxybenzoic hydroxylase
-
-
-
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para-hydroxybenzoate hydroxylase
-
-
-
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PHBAD
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-
-
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PHBH
PHBHase
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-
-
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POHBase
-
-
-
-
p-hydroxybenzoate hydroxylase
-
-
-
-
PHBH
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-hydroxybenzoate + NADPH + H+ + O2 = 3,4-dihydroxybenzoate + NADP+ + H2O
bi uni uni uni ping-pong mechanism
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
-
-
-
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reduction
-
-
-
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PATHWAY SOURCE
PATHWAYS
-
-, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating)
A flavoprotein (FAD). Most enzymes from Pseudomonas are highly specific for NADPH (cf. EC 1.14.13.33 4-hydroxybenzoate 3-monooxygenase [NAD(P)H]).
CAS REGISTRY NUMBER
COMMENTARY
9059-23-8
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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-hydroxybenzoate + NADPH + H+ + O2
3,4-dihydroxybenzoate + NADP+ + H2O
-
-
-
ir
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
3-bromo-4-hydroxybenzoate + NADPH + O2
?
-
3.2% of the activity with 4-hydroxybenzoate
-
-
?
3-Fluoro-4-hydroxybenzoate + NADPH + O2
?
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
4-aminobenzoate + NADPH + O2
?
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + ferricyanide
protocatechuate + NADP+ + ferrocyanide
-
-
-
?
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
4-mercaptobenzoate + NADPH + O2
4,4'-dithiobisbenzoate + ?
-
50% of the activity with 4-hydroxybenzoate
-
?
4-toluate + NADPH + O2
?
-
0.29% of the activity with 4-hydroxybenzoate
-
-
?
benzene sulfonate + NADPH + O2
?
-
0.34% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
-
-
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
-
1.5% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
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slow reaction, formation of at least 3 intermediates, a spectral intermediate that is believed to be an oxygenated form of the enzyme-bound flavin prosthetic group
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
inducible enzyme
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
first step in the bacterial metabolism when 4-hydroxybenzoate is used as growth substrate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
enzyme catalyzes an intermediate step in the degradation of aromatic compounds in soil microorganisms
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
390020, 390021, 390022, 390023, 390025, 390027, 390030, 390032, 390033, 390034, 390041, 390042, 390044, 390046, 390049, 686087
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
at least three intermediates
-
-
?
additional information
?
-
development of a colorimetric coupled assay: the glucose-6-phosphate dehydrogenase (G6PD) catalyzes the specific dehydrogenation of glucose-6-phosphate (G-6-P) by consuming the oxidized form of beta-nicotinamide adenine dinucleotide phosphate (NADP+) to generate NADPH. And the formed NADPH initiates the irreversible hydroxylation of p-hydroxybenzoate by p-hydroxybenzoate hydroxylase (PHBH) to generate 3,4-dihydroxybenzoate (DHB), which results in the subsequent in situ generation of a photoresponsive nanozyme of DHB coordinated SrTiO3 (SrTiO3/DHB) nanosheets. The photoresponsive nanozyme has intriguing interfacial charge transfer (ICT) characteristics and thus realizes the efficiently catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under light irradiation for signal readout. Optimization and evaluation of the ultrasensitive colorimetric bioassay for versatile targets, overview
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-
-
additional information
?
-
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Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH
-
-
?
additional information
?
-
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under anaerobic conditions, the enzyme can catalyze a reduction of FAD by NADPH provided that 4-hydroxybenzoate is present
-
-
?
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-hydroxybenzoate + NADPH + H+ + O2
3,4-dihydroxybenzoate + NADP+ + H2O
-
-
-
ir
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
inducible enzyme
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
first step in the bacterial metabolism when 4-hydroxybenzoate is used as growth substrate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
-
enzyme catalyzes an intermediate step in the degradation of aromatic compounds in soil microorganisms
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
arabinoflavin adenine dinucleotide
-
like native enzyme the arabinoflavin adenine dinucleotide containing 4-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate. The oxidative part of the catalytic cycle of a FAD-containing 4-hydroxybenzoate hydroxylase differs from the native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of H2O2 per mol of NADPH oxidized
FAD
-
flavin motion in 4-hydroxybenzoate hydroxylase is important for efficient reduction
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diethyl dicarbonate
-
inhibition of wild-type enzyme, no inhibition of mutant enzyme H162R
additional information
-
one of the five sulfhydryl groups reacts rapidly and specifically with NEM, without inactivation of the enzyme
-
additional information
-
multisubunit inhibition by ether derivatives
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2,4-Dihydroxybenzoate
-
increases the rate of NADP oxidation by 4-hydroxybenzoate, no hydroxylation during subsequent reoxidation by O2
3,4-dihydroxybenzoate
-
increases the rate of NADP oxidation by 4-hydroxybenzoate, no hydroxylation during subsequent reoxidation by O2
benzoate
-
increases the rate of NADP oxidation by 4-hydroxybenzoate, no hydroxylation during subsequent reoxidation by O2
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.03
4-hydroxybenzoate
-
mutant enzyme H162R, H162Y, H162K, R269K, R269Y, R269N, R269S and R269T
additional information
additional information
-
Km value for NADPH above 0.5 mM: mutant enzymes R269D, R269T, R269N, R269Y, H162D, H162T, H162S, H162N
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
monomer
-
the monomeric form of PHBH, which is not achieved under conventional conditions, is isolated by entrapment in reverse micelles and by addition of DMSO. The PHBH monomer is catalytically more efficient than the PHBH dimer
dimer
-
the monomeric form of PHBH, which is not achieved under conventional conditions, is isolated by entrapment in reverse micelles and by addition of DMSO. The PHBH monomer is catalytically more efficient than the PHBH dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal of the enzyme complexed with 4-hydroxybenzoate are obtained using the hanging-drop method
-
crystal structure of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate
-
crystallization of mutant enzymes H162R and R269T by hanging drop vapour diffusion method
-
crystals of a arabinoflavin adenine dinucleotide -containing 4-hydroxybenzoate hydroxylase in complex with 4-hydroxybenzoate are obtained using the hanging drop method
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H162K
-
less efficient than wild-type enzyme due to a clear increase in the apparent Km-value for NADPH
H162R
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
H162Y
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
R269K
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
R269S
-
less efficient than wild-type enzyme due to a clear increase in the apparent Km-value for NADPH
Y222A
Y222V
Y222A
-
mutation makes the lifetime distribution of FAD in the enzyme simpler by removing the ultrafast 10-15 ps lifetime component
Y222V
-
mutation makes the lifetime distribution of FAD in the enzyme simpler by removing the ultrafast 10-15 ps lifetime component
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
microheterogeneity of the highly purified enzyme. Different form of enzyme molecules are due to the partial oxidation of Cys116 in the sequence of the enzyme
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant enzymes R42S and R42K expressed in transformed Escherichia coli TG2 cells
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Howell, L.G.; Spector, T.; Massey, V.
Purification and properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens
J. Biol. Chem.
247
4340-4350
1972
Pseudomonas fluorescens
Spector, T.; Massey, V.
Studies on the effector specificity of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens
J. Biol. Chem.
247
4679-4687
1972
Pseudomonas fluorescens
Spector, T.; Massey, V.
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Evidence for an oxygenated flavin intermediate
J. Biol. Chem.
247
5632-5636
1972
Pseudomonas fluorescens
Hosokawa, K.; Stanier, R.Y.
Crystallization and properties of p-hydroxybenzoate hydroxylase from Pseudomonas putida
J. Biol. Chem.
241
2453-2460
1966
Pseudomonas fluorescens
Steennis, P.J.; Cordes, M.M.; Hilkens, J.G.H.; Mueller, F.
On the interaction of para-hydroxybenzoate hydroxylase from Pseudomonas fluorescens with halogen ions
FEBS Lett.
36
177-180
1973
Pseudomonas fluorescens
Husain, M.; Schopfer, L.M.; Massey, V.
p-Hydroxybenzoate hydroxylase and melilotate hydroxylase
Methods Enzymol.
53
543-558
1978
Delftia acidovorans, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas putida A 3.12, Pseudomonas putida M-6
Drenth, J.; Hol, W.G.J.; Wierenga, R.K.
Crystallization and preliminary x-ray investigation of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens
J. Biol. Chem.
250
5268-5269
1975
Pseudomonas fluorescens
van Berkel, W.J.H.; Mueller, F.
The temperature and pH dependence of some properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Eur. J. Biochem.
179
307-314
1989
Pseudomonas fluorescens
Entsch, B.; Ballou, D.P.; Massey, V.
Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase
J. Biol. Chem.
251
2550-2563
1976
Pseudomonas fluorescens
van Berkel, W.J.H.; Mueller, F.
The elucidation of the microheterogeneity of highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques
Eur. J. Biochem.
167
35-46
1987
Pseudomonas fluorescens
Mueller, F.; Voordouw, G.; van Berkel, W.J.H.; Steennis, P.J.; Visser, S.; van Rooijen, P.J.
A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Improved purification, relative molecular mass, and amino acid composition
Eur. J. Biochem.
101
235-244
1979
Pseudomonas fluorescens
van Berkel, W.J.H.; Weijer, W.J.; Mueller, F.; Jekel, P.A.; Beintema, J.J.
Chemical modification of sulfhydryl groups in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Involvement in catalysis and assignment in the sequence
Eur. J. Biochem.
145
245-256
1984
Pseudomonas fluorescens
Schreuder, H.A.; van der Laan, J.M.; Hol, W.G.J.; Drenth, J.
Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate
J. Mol. Biol.
199
637-648
1988
Pseudomonas fluorescens
Salituro, F.G.; Demeter, D.A.; Weintraub, H.J.R.; Lippert, B.J.; Resvick, R.J.; McDonald, I.A.
Multisubstrate inhibition of 4-hydroxybenzoate 3-monooxygenase
J. Med. Chem.
37
4076-4078
1994
Pseudomonas fluorescens
van Berkel, W.J.H.; Eppink, M.H.M.; Schreuder, H.A.
Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin
Protein Sci.
3
2245-2253
1994
Pseudomonas fluorescens
Abe, I.; Kashiwagi, K.; Noguchi, H.
Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase
FEBS Lett.
483
131-134
2000
Pseudomonas aeruginosa, Pseudomonas fluorescens (P00438)
Eppink, M.H.M.; Schreuder, H.A.; Van Berkel, W.J.H.
Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding
Eur. J. Biochem.
253
194-201
1998
Pseudomonas fluorescens
Eppink, M.H.M.; Schreuder, H.A.; Van Berkel, W.J.H.
Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants
J. Biol. Chem.
273
21031-21039
1998
Pseudomonas fluorescens
Schreuder, H.A.; Mattevi, A.; Obmolova, G.; Kalk, K.H.; Hol, W.G.J.; van der Bolt, F.J.T.; van Berkel, W.J.H.
Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring
Biochemistry
33
10161-10170
1994
Pseudomonas fluorescens
Kudryashova, E.V.; Visser, A.J.; van Berkel, W.J.
Monomer formation and function of p-hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide/water mixtures
Chembiochem
9
413-419
2008
Pseudomonas fluorescens
Van Den Berg, P.A.; Grever, K.; Van Hoek, A.; Van Berkel, W.J.; Visser, A.J.
Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase
J. Chem. Sci.
119
123-133
2007
Pseudomonas fluorescens
-
Van Den Berg, P.A.; Grever, K.; Van Hoek, A.; Van Berkel, W.J.; Visser, A.J.
Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase
J. Chem. Sci.
119
123-133
2007
Pseudomonas fluorescens
-
Sun, D.; Liu, T.; Dong, Y.; Liu, Q.; Wu, X.; Wang, G.
Coupling p-hydroxybenzoate hydroxylase with the photoresponsive nanozyme for universal dehydrogenase-based bioassays
Sens. Actuators B Chem.
327
128859
2021
Pseudomonas fluorescens (P00438), Pseudomonas fluorescens Migula 1895 (P00438)
-
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