Information on EC 1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase

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

EC NUMBER
COMMENTARY
1.14.13.2
-
RECOMMENDED NAME
GeneOntology No.
4-hydroxybenzoate 3-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4-hydroxybenzoate + NADPH + H+ + O2 = protocatechuate + NADP+ + H2O
show the reaction diagram
bi uni uni uni ping-pong mechanism
-
4-hydroxybenzoate + NADPH + H+ + O2 = protocatechuate + NADP+ + H2O
show the reaction diagram
rate of formation of the flavin hydroperoxide is not influenced by pH-change. Rate of hydroxylation reaction increases with pH. The H-bond network abstracts the phenolic proton from p-hydroxybenzoate in the transition state of oxygen transfer. Product deprotonation enhances the rate of a specific conformational change required for both product relase and the elimination of water
-
4-hydroxybenzoate + NADPH + H+ + O2 = protocatechuate + NADP+ + H2O
show the reaction diagram
kinetic scheme of reductive half-reaction, flavin movement can occur in the presence or absence of NADPH, but NADPH stimulates movement to the reactive conformation required for hydride transfer
-
4-hydroxybenzoate + NADPH + H+ + O2 = protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
4-chlorobenzoate degradation
-
4-hydroxymandelate degradation
-
Benzoate degradation
-
Metabolic pathways
-
Microbial metabolism in diverse environments
-
toluene degradation to protocatechuate (via p-cresol)
-
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]).
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4-HBA 3-monooxygenase
-
-
4-HBA-3-hydroxylase
-
-
-
-
4-hydroxybenzoate 3-hydroxylase
-
-
-
-
4-hydroxybenzoate 3-hydroxylase
-
-
4-hydroxybenzoate 3-hydroxylase
-
-
-
4-hydroxybenzoate 3-hydroxylase
-
-
4-hydroxybenzoate 3-hydroxylase
Pseudomonas sp. PPD
-
-
-
4-hydroxybenzoate 3-monooxygenase
-
-
-
-
4-hydroxybenzoate hydroxylase
-
-
4-hydroxybenzoate monooxygenase
-
-
-
-
4-hydroxybenzoic hydroxylase
-
-
-
-
4HBA 3-hydroxylase
-
-
BxeA2040
-
gene name
m-hydroxybenzoate hydroxylase
Q6SSJ6
-
m-hydroxybenzoate hydroxylase
Comamonas testosteroni GZ39
Q6SSJ6
-
-
MobA
Comamonas testosteroni GZ39
Q6SSJ6
-
-
oxygenase, 4-hydroxybenzoate 3-mono-
-
-
-
-
p-hydroxybenzoate hydroxylase
-
-
-
-
p-hydroxybenzoate hydroxylase
-
-
p-hydroxybenzoate hydroxylase
Acinetobacter baylyi ADPU54, Acinetobacter baylyi Crc
-
-
-
p-hydroxybenzoate hydroxylase
-
-
p-hydroxybenzoate hydroxylase
-
-
-
p-hydroxybenzoate hydroxylase
-
-
p-hydroxybenzoate hydroxylase
Q9R9T1
-
p-hydroxybenzoate hydroxylase
Pseudomonas putida WCS358
Q9R9T1
-
-
p-hydroxybenzoate hydroxylase
-
-
p-hydroxybenzoate-3-hydroxylase
-
-
-
-
p-hydroxybenzoic acid hydrolase
-
-
-
-
p-hydroxybenzoic acid hydroxylase
-
-
-
-
p-hydroxybenzoic acid hydroxylase
Q9R9T1
-
p-hydroxybenzoic acid hydroxylase
Pseudomonas putida WCS358
Q9R9T1
-
-
p-hydroxybenzoic hydroxylase
-
-
-
-
para-hydroxybenzoate hydroxylase
-
-
-
-
PHBAD
-
-
-
-
PHBH
-
-
-
-
PHBHase
-
-
-
-
PobA
Q9R9T1
-
PobA
Pseudomonas putida WCS358
Q9R9T1
-
-
POHBase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9059-23-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain ADPU54
-
-
Manually annotated by BRENDA team
Acinetobacter baylyi ADPU54
strain ADPU54
-
-
Manually annotated by BRENDA team
strain PPH
-
-
Manually annotated by BRENDA team
strain PPH
-
-
Manually annotated by BRENDA team
strain HS-2
-
-
Manually annotated by BRENDA team
Comamonas testosteroni GZ39
-
UniProt
Manually annotated by BRENDA team
Comamonas testosteroni Kh 122-3S
Kh 122-3S
-
-
Manually annotated by BRENDA team
enzyme is expressed in mutant strain MAO4, but not in wild-type strain
-
-
Manually annotated by BRENDA team
strain GU2
-
-
Manually annotated by BRENDA team
Moraxella sp. GU2
strain GU2
-
-
Manually annotated by BRENDA team
Phomopsis liquidambaris B3
-
-
-
Manually annotated by BRENDA team
Pseudomonas mendocina KR1
KR1
-
-
Manually annotated by BRENDA team
A 3.12; M-6
-
-
Manually annotated by BRENDA team
strain WCS358
UniProt
Manually annotated by BRENDA team
Pseudomonas putida A 3.12
A 3.12
-
-
Manually annotated by BRENDA team
Pseudomonas putida M-6
M-6
-
-
Manually annotated by BRENDA team
Pseudomonas putida WCS358
strain WCS358
UniProt
Manually annotated by BRENDA team
strain PPD
-
-
Manually annotated by BRENDA team
Pseudomonas sp. PPD
strain PPD
-
-
Manually annotated by BRENDA team
557, 420, and 1G
-
-
Manually annotated by BRENDA team
Rhodococcus rhodnii 135
135
-
-
Manually annotated by BRENDA team
Rhodococcus rhodochrous 172
172
-
-
Manually annotated by BRENDA team
Rhodococcus sp. 400
400
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
the pobA gene encoding the 4-hydroxybenzoate 3-monooxygenase is expressed during growth on hydroxybenzoic acids and glucose
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,3-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
2,3-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
Comamonas testosteroni GZ39
Q6SSJ6
-
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
P20586
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
3.1% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
1.5% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
8% of the activity with 4-hydroxybenzoate
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
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
-
-
?
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
show the reaction diagram
-
8% of the activity with 4-hydroxybenzoate
-
-
?
2,5-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
2,5-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
Comamonas testosteroni GZ39
Q6SSJ6
-
-
-
?
2-chloro-4-hydroxybenzoate + NADH + O2
?
show the reaction diagram
-
40% of the activity with 4-hydroxybenzoate
-
-
?
2-fluoro-4-hydroxybenzoate + NADH + O2
?
show the reaction diagram
-
50% of the activity with 4-hydroxybenzoate
-
-
?
3,4-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
3,4-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
Comamonas testosteroni GZ39
Q6SSJ6
-
-
-
?
3,5-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
3,5-dihydroxybenzoate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
Comamonas testosteroni GZ39
Q6SSJ6
-
-
-
?
3-bromo-4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
3.2% of the activity with 4-hydroxybenzoate
-
-
?
3-Chloro-4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
6.5% of the activity with 4-hydroxybenzoate
-
-
?
3-chlorophenol + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
3-Fluoro-4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
3-hydroxyanthranilate + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
4-aminobenzoate + NADPH + O2
?
show the reaction diagram
-
about 1% of the activity with 4-hydroxybenzoate
-
-
?
4-chlorophenol + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
4-chlororesorcinol + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
the enzyme prefers NADPH to NADH
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
Rhodococcus rhodnii 135, Rhodococcus rhodochrous 172
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
Moraxella sp. GU2
-
-
-
-
?
4-hydroxybenzoate + NADH + O2
protocatechuate + NAD+ + H2O
show the reaction diagram
Rhodococcus sp. 400
-
-
-
?
4-hydroxybenzoate + NADPH + ferricyanide
protocatechuate + NADP+ + ferrocyanide
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Phomopsis liquidambaris, Phomopsis liquidambaris B3
-
4-hydroxybenzoate degradation proceeds via hdroxylation to 3,4-dihydroxybenzoic acid and then conversion to catechol, which is cleaved to cis,cis-muconic acid through ortho-catechol cleavage
4-hydroxybenzoate degradation proceeds via hdroxylation to 3,4-dihydroxybenzoic acid and then conversion to catechol, which is cleaved to cis,cis-muconic acid through ortho-catechol cleavage
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
P00438
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
-
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
P20586
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Q9R9T1
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
at least three intermediates
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
the enzyme prefers NADPH to NADH. 4HBA hydroxylase can be induced by 4HBA, 4-cresol, and 4-hydroxybenzaldehyde, the enzyme prefers NADPH to NADH
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas putida A 3.12
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Comamonas testosteroni Kh 122-3S
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Rhodococcus rhodnii 135
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas putida WCS358
Q9R9T1
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas putida M-6
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-, enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Rhodococcus rhodochrous 172
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Moraxella sp. GU2
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas sp. PPD
-
-, enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Rhodococcus sp. 400
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas mendocina KR1
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
inducible enzyme
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
first step in the bacterial metabolism when 4-hydroxybenzoate is used as growth substrate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme of the toluene-4-monooxygenase catabolic pathway
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme catalyzes an intermediate step in the degradation of aromatic compounds in soil microorganisms
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
degradation of 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme is expressed at basal level, presence of 4-hydroxybenzoate enhances activity
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Comamonas testosteroni Kh 122-3S
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Moraxella sp. GU2
-
degradation of 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Pseudomonas mendocina KR1
-
enzyme of the toluene-4-monooxygenase catabolic pathway
-
-
-
4-mercaptobenzoate + NADPH + O2
4,4'-dithiobisbenzoate + ?
show the reaction diagram
-
-
-
-
?
4-mercaptobenzoate + NADPH + O2
4,4'-dithiobisbenzoate + ?
show the reaction diagram
-
50% of the activity with 4-hydroxybenzoate
-
?
4-nitrophenol + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
4-toluate + NADPH + O2
?
show the reaction diagram
-
0.29% of the activity with 4-hydroxybenzoate
-
-
?
benzene sulfonate + NADPH + O2
?
show the reaction diagram
-
0.34% of the activity with 4-hydroxybenzoate
-
-
?
hydroquinone + NADP+ + O2
? + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
p-hydroxybenzoate + NADPH + O2
? + NAD+ + H2O
show the reaction diagram
-, Q6SSJ6
is transformed to a lesser extent than m-hydroxybenzoate
-
-
?
m-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-, Q6SSJ6
-
-
-
?
additional information
?
-
-
mechanism of oxygen insertion
-
-
-
additional information
?
-
-
a Tyr seems to be involved in substrate activation
-
-
-
additional information
?
-
-
Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH
-
-
-
additional information
?
-
-
mutant enzyme Y385F hydroxylates 3,4-dihydroxybenzoate to form gallic acid
-
-
-
additional information
?
-
-
under anaerobic conditions, the enzyme can catalyze a reduction of FAD by NADPH provided that 4-hydroxybenzoate is present
-
-
-
additional information
?
-
-
p-hydroxybenzoate induces the expression of p-hydroxybenzoate hydroxylase
-
-
-
additional information
?
-
-
no hydroxylase activity is observed with 3-hydroxybenzoate, resorcinol, 3-nitrophenol, benzoate, 2,4-dihydroxybenzoate, 4-chlorobenzoate, 4-aminobenzoate, 4-aminophenol, 4-nitrophenol, and 4-dinitrobenzene
-
-
-
additional information
?
-
-, Q6SSJ6
is not capable of hydroxylating benzoate, o-hydroxybenzoate (salicylate), 2,4-dihydroxybenzoate, 2,6-dihydroxybenzoate, 2-chlorophenol, 3-aminophenol, 4-methoxybenzoate, 3-toluate, o-cresol, m-cresol, or p-cresol
-
-
-
additional information
?
-
Comamonas testosteroni GZ39
Q6SSJ6
is not capable of hydroxylating benzoate, o-hydroxybenzoate (salicylate), 2,4-dihydroxybenzoate, 2,6-dihydroxybenzoate, 2-chlorophenol, 3-aminophenol, 4-methoxybenzoate, 3-toluate, o-cresol, m-cresol, or p-cresol
-
-
-
additional information
?
-
-
p-hydroxybenzoate induces the expression of p-hydroxybenzoate hydroxylase
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Phomopsis liquidambaris, Phomopsis liquidambaris B3
-
4-hydroxybenzoate degradation proceeds via hdroxylation to 3,4-dihydroxybenzoic acid and then conversion to catechol, which is cleaved to cis,cis-muconic acid through ortho-catechol cleavage
4-hydroxybenzoate degradation proceeds via hdroxylation to 3,4-dihydroxybenzoic acid and then conversion to catechol, which is cleaved to cis,cis-muconic acid through ortho-catechol cleavage
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
the enzyme prefers NADPH to NADH. 4HBA hydroxylase can be induced by 4HBA, 4-cresol, and 4-hydroxybenzaldehyde
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
inducible enzyme
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
first step in the bacterial metabolism when 4-hydroxybenzoate is used as growth substrate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme of the toluene-4-monooxygenase catabolic pathway
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme catalyzes an intermediate step in the degradation of aromatic compounds in soil microorganisms
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
-
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
degradation of 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
-
enzyme is expressed at basal level, presence of 4-hydroxybenzoate enhances activity
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Comamonas testosteroni Kh 122-3S
-
high degree of homology observed between the enzyme from Comamonas and of Pseudomonas and Acinetobacter indicates the common evolutionary origin of the enzyme in the divergent pathways of 4-hydroxybenzoate among these soil bacteria of different genera
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
-
enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Moraxella sp. GU2
-
degradation of 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
show the reaction diagram
Pseudomonas sp. PPD
-
enzyme is induced by 4-hydroxybenzoate
-
-
?
4-hydroxybenzoate + NADPH + O2
?
show the reaction diagram
Pseudomonas mendocina KR1
-
enzyme of the toluene-4-monooxygenase catabolic pathway
-
-
-
additional information
?
-
-
p-hydroxybenzoate induces the expression of p-hydroxybenzoate hydroxylase
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1-deaza-FAD
-
when 1-deaza-FAD is used as cofactor, the enzyme carries out each step in catalysis except the transfer of oxygen to 4-hydroxybenzoate
6-Hydroxy-FAD
-
when 6-hydroxy-FAD is used as cofactor, the enzyme has a lower turnover rate than the native enzyme
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
-
contains approximately 0.8 mol FAD per mol of enzyme
FAD
-
contains 1 mol of FAD per mol of enzyme
FAD
-
contains 1 mol of FAD per subunit
FAD
-
contains 1 mol of FAD per mol of enzyme
FAD
-
Km for strain 420: 220 nM; Km for strain 557: 190 nM; Km for strain IG: 185 nM
FAD
-
Km: 440 nM
FAD
-
movement of the flavin occurs before reduction
FAD
-
contains 0.12 mol FAD per mol of enzyme
FAD
-
flavin motion in 4-hydroxybenzoate hydroxylase is important for efficient reduction
FAD
-
Km: 150 nM
FAD
-
thermodynamic and kinetic constants of the enzyme reconstituted with 8-substituted flavins; two flavin conformations in the enzyme: the in-position and the out-position. Substrate hydroxylation occurs while the flavin in the enzyme is in the in-conformation. Flavin must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction
NADH
-
activity with NADH is higher than activity with NADPH
NADH
-
activity with NADH is higher than activity with NADPH
NADH
-
the enzyme prefers NADPH to NADH
NADPH
-
no activity with NADH
NADPH
-
no activity with NADH
NADPH
-
activity with NADH is higher than activity with NADPH
NADPH
-
the enzyme prefers NADPH to NADH
additional information
-
helix H2 is involved in determining the coenzyme specificity
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
metal ions (at 1 mM) Co2+, Ni2+, Mg2+, and Hg2+ shows little influence on hydroxylase activity
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(-)-epigallocatechin-3-O-gallate
-
non-competitive, binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds
2,4-Dihydroxybenzoate
-
competitive with 4-hydroxybenzoate
3,4-dihydroxybenzoate
-
at high concentration
3,4-dihydroxybenzoate
-
competitive with 4-hydroxybenzoate
3-Chloro-4-hydroxybenzoate
-
mixed-type
3-Hydroxybenzoate
-
above 1 mM
3-Hydroxybenzoate
-
2 mM, 26% inhibition
4-Aminobenzoate
-
above 1 mM
4-Aminobenzoate
-
competitive
4-Aminosalicylate
-
competitive
4-Fluorobenzoate
-
slight
4-Fluorobenzoate
-
above 1 mM
4-hydroxy-3-nitrobenzoic acid
-
2 mM, 23% inhibition
4-hydroxybenzaldehyde
-
slight
4-hydroxybenzaldehyde
-
2 mM, 27% inhibition
4-hydroxybenzoate
-
at high concentrations
4-hydroxybenzoate
-
above 1 mM
4-hydroxybenzoate
-
mixed-type
4-hydroxybenzoate
-
above 1 mM
4-hydroxybenzoate
-
above 0.1 mM
4-hydroxybutyrate
-
92 mM, pH 8, very slight inhibition
4-hydroxycinnamate
-
-
4-hydroxyphenylacetic acid
-
2 mM, 16% inhibition
4-nitrophenol
-
2 mM, 39% inhibition
6-Aminonicotinate
-
competitive
6-Hydroxynicotinate
-
at high concentrations
6-Hydroxynicotinate
-
-
acetate
-
92 mM, pH 8, very slight inhibition
Benzoate
-
above 1 mM
Benzoate
-
competitive with 4-hydroxybenzoate
Benzoate
-
2 mM, 48% inhibition
Br-
-
competitive with respect to NADPH
Br-
-
competitive with respect to NADPH; mixed type inhibition with respect to 4-hydroxybenzoate
citrate
-
92 mM, pH 8, very slight inhibition
Cl-
-
competitive with respect to NADPH
Cl-
-
competitive with respect to NADPH; mixed type inhibition with respect to 4-hydroxybenzoate
Cl-
-
non-competitive with NADPH
Cl-
-
competitive with respect to NADH
Cl-
-
50 mM, significant inhibition. No inhibition at 5 mM
CNS-
-
competitive with respect to NADPH
coumaric acid
-
2 mM, 30% inhibition
Cu2+
-
1 mM, moderate inhibition
diethyl dicarbonate
-
-
diethyl dicarbonate
-
inhibition of wild-type enzyme, no inhibition of mutant enzyme H162R
F-
-
competitive with respect to NADPH
Fe3+
-
1 mM, moderate inhibition
-
formate
-
92 mM, pH 8, very slight inhibition
fumarate
-
92 mM, pH 8, very slight inhibition
Hg2+
-
0.1 mM HgCl2, complete inhibition
I-
-
competitive with respect to NADPH
I-
-
competitive with respect to NADPH; mixed type inhibition with respect to 4-hydroxybenzoate
iodoacetamide
-
reversed by dithiothreitol
Maleate
-
92 mM, pH 8, very slight inhibition
-
n-dodecyl gallate
-
non-competitive
N-iodosuccinimide
-
reversed by dithiothreitol
NEM
-
0.1 mM, 59% inhibition
o-Iodosobenzoate
-
reversed by dithiothreitol
p-hydroxy-3-iodomethylbenzoate
-
1 mM, irreversible crosslinking to the substrate binding site
PCMB
-
0.015 mM, more than 70% inhibition, partially restored by addition of 0.14 M 2-mercaptoethanol
Phenylglyoxal
-
pseudo-first order kinetics, incorporation into the substrate-binding site
phosphate
-
competitive inhibition
Propionate
-
92 mM, pH 8, very slight inhibition
protocatechuate
-
above 1 mM
salicylate
-
above 1 mM
Tartrate
-
92 mM, pH 8, very slight inhibition
Zn2+
-
0.2 mM, reversed by 10 mM glutathione
Zn2+
-
1 mM, moderate inhibition
Mn2+
-
1 mM, moderate inhibition
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
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
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
5-Hydroxypicolinate
-
stimulates rapid oxidation of NADPH
Benzoate
-
increases the rate of NADP oxidation by 4-hydroxybenzoate, no hydroxylation during subsequent reoxidation by O2
dithiothreitol
-
4 mM, slightly increases activity
EDTA
-
5 mM or 20 mM, slightly increases activity
additional information
-
the substrate analogue 5-hydroxypicolinate stimulates high rates of NADPH consumption by PHBH without the formation of an oxygenated product of 5-hydroxypicolinate
-
additional information
-
transcript levels of pobA, encoding p-hydroxybenzoate hydroxylase, are slightly increased in the absence of catabolite repression control protein Crc
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.014
-
2,4-Dihydroxybenzoate
-
-
0.25
-
2,4-Dihydroxybenzoate
-
-
0.18
-
3-Chloro-4-hydroxybenzoate
-
-
0.0006
-
4-hydroxybenzoate
-
pH 8, monomeric form
0.003
-
4-hydroxybenzoate
-
strain 557
0.0033
-
4-hydroxybenzoate
-
-
0.0072
-
4-hydroxybenzoate
-
-
0.0073
-
4-hydroxybenzoate
-
-
0.0079
-
4-hydroxybenzoate
-
strain IG
0.008
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38Y/T42R
0.0081
-
4-hydroxybenzoate
-
strain 420
0.0095
-
4-hydroxybenzoate
-
reaction with NADH
0.0109
-
4-hydroxybenzoate
-
-
0.012
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38A
0.014
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme T42R
0.02
-
4-hydroxybenzoate
-
wild-type enzyme
0.0213
-
4-hydroxybenzoate
-
-
0.022
-
4-hydroxybenzoate
-
-
0.025
-
4-hydroxybenzoate
-
wild-type enzyme
0.025
-
4-hydroxybenzoate
-
pH 8, dimeric form
0.026
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38Y
0.03
-
4-hydroxybenzoate
-
mutant enzyme H162R, H162Y, H162K, R269K, R269Y, R269N, R269S and R269T
0.036
-
4-hydroxybenzoate
-
pH 8.0, 25C, wild-type enzyme
0.04
-
4-hydroxybenzoate
-
mutant enzyme H162N, H162S, H162T and H162D
0.041
-
4-hydroxybenzoate
-
-
0.06
-
4-hydroxybenzoate
-
mutant enzyme R269D
0.075
-
4-hydroxybenzoate
-
mutant enzyme R42S
0.075
-
4-hydroxybenzoate
-
35C
0.0122
-
NADH
-
strain 557
0.0139
-
NADH
-
-
0.0195
-
NADH
-
strain IG
0.02
-
NADH
-
strain 420
0.037
-
NADH
-
-
0.0398
-
NADH
-
-
0.08
-
NADH
-
25C
0.12
-
NADH
-
35C
0.18
-
NADH
-
45C
0.027
-
NADP+
-
reaction with 2,4-dihydroxybenzoate
0.0227
-
NADPH
-
reaction with 4-hydroxybenzoate
0.0238
-
NADPH
-
strain IG
0.025
-
NADPH
-
pH 8, dimeric form
0.03
-
NADPH
-
wild-type enzyme
0.0335
-
NADPH
-
strain 557
0.04
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38A
0.045
-
NADPH
-
pH 8.0, 25C, mutant enzyme T42R
0.047
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38Y
0.05
-
NADPH
-
wild-type enzyme
0.05
-
NADPH
-
mutant enzyme H162R
0.052
-
NADPH
-
pH 8, monomeric form
0.0658
-
NADPH
-
strain 420
0.07
-
NADPH
-
mutant enzyme H162Y and R269K
0.084
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38Y/T42R
0.14
-
NADPH
-
25C
0.155
-
NADPH
-
-
0.16
-
NADPH
-
35C
0.195
-
NADPH
-
pH 8.0, 25C, wild-type enzyme
0.2
-
NADPH
-
at 45C
0.2
-
NADPH
-
mutant enzyme H162K
0.32
-
NADPH
-
mutant enzyme R269S
0.05
-
O2
-
reaction with 2,4-dihydroxybenzoate
0.11
-
4-hydroxybenzoate
-
mutant enzyme R42K
additional information
-
additional information
-
-
-
additional information
-
additional information
-
-
-
additional information
-
additional information
-
Km value for NADPH above 0.5 mM: mutant enzymes R269D, R269T, R269N, R269Y, H162D, H162T, H162S, H162N
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.36
-
4-hydroxybenzoate
-
mutant enzyme Y385F
0.42
-
4-hydroxybenzoate
-
mutant enzyme Y201F
2.8
-
4-hydroxybenzoate
-
pH 8.0, 25C, wild-type enzyme
5.7
-
4-hydroxybenzoate
-
wild-type enzyme
7.4
-
4-hydroxybenzoate
-
35C
7.5
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme T42R
15.2
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38A
20.4
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38Y/T42R
24.3
-
4-hydroxybenzoate
-
pH 8.0, 25C, mutant enzyme D38Y
36.7
-
4-hydroxybenzoate
-
-
45
-
4-hydroxybenzoate
-
pH 8, monomeric form
6.8
-
NADH
-
25C
11.6
-
NADH
-
35C
16.9
-
NADH
-
45C
6.6
-
NADPH
-
25C
7.8
-
NADPH
-
pH 8.0, 25C, wild-type enzyme
8.6
-
NADPH
-
35C
12.4
-
NADPH
-
pH 8.0, 25C, mutant enzyme T42R
13.3
-
NADPH
-
45C
18.8
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38A
19.6
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38Y
45
-
NADPH
-
pH 8, monomeric form
46.4
-
NADPH
-
pH 8.0, 25C, mutant enzyme D38Y/T42R
63
-
NADPH
-
pH 8, dimeric form
0.2
-
p-hydroxybenzoate
-
4C, pH 6.5, E49Q mutant
1.5
-
p-hydroxybenzoate
-
4C, pH 8.6, E49Q mutant
6.2
-
p-hydroxybenzoate
-
4C, pH 6.5, wild-type PHBH
7.5
-
p-hydroxybenzoate
-
4C, pH 8.6, wild-type PHBH
63
-
4-hydroxybenzoate
-
pH 8, dimeric form
additional information
-
additional information
-
-
-
additional information
-
additional information
-
-
-
additional information
-
additional information
-
-
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.014
-
(-)-epigallocatechin-3-O-gallate
-
-
0.31
-
2,4-Dihydroxybenzoate
-
-
0.55
-
3,4-dihydroxybenzoate
-
-
0.05
-
4-Aminobenzoate
-
-
0.0181
-
n-dodecyl gallate
-
-
4.3
-
phosphate
-
4C, pH 6.8
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
10.2
-
-
-
additional information
-
-
-
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
7.3
-
reaction with NADH
7.2
-
-
strain 400 and 557
7.4
-
-
strain IG
7.5
7.8
-
reaction with NADPH
8
8.1
-
HEPES or Tris-SO42- buffer
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
PDB
SCOP
CATH
ORGANISM
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
44000
-
Q9R9T1
estimated from amino acid sequence
65000
-
-
gel filtration
75000
-
-
gel filtration
76000
82000
-
gel filtration
78000
-
-
gel filtration
80000
-
-
gel filtration
80000
-
-
gel filtration, non-denaturing PAGE
83000
90000
-
equilibrium sedimentation
83600
-
-
low speed sedimentation without reaching equilibrium
85000
-
-
gel filtration
89000
-
-
gel filtration
90000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 71000, SDS-PAGE
dimer
-
2 * 45100
dimer
-
2 * 45000, SDS-PAGE
dimer
-
1 * 43000-45000, SDS-PAGE
dimer
-
2 * 40000, SDS-PAGE
dimer
-
2 * 45000, SDS-PAGE
dimer
-
2 * 43632, calculation from nucleotide sequence; 2 * 44000, SDS-PAGE
dimer
-
2 * 45000, SDS-PAGE
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
dimer
Moraxella sp. GU2
-
2 * 45000, SDS-PAGE
-
dimer
-
2 * 43632, calculation from nucleotide sequence; 2 * 44000, SDS-PAGE
-
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
tetramer
-
4 * 46000, strain 557, SDS-PAGE; 4 * 47500, strain 420, SDS-PAGE; 4 * 51000, strain IG, SDS-PAGE
tetramer
-
4 * 48500, SDS-PAGE
tetramer
-
4 * 45000, SDS-PAGE
tetramer
Rhodococcus rhodnii 135
-
4 * 48500, SDS-PAGE
-
tetramer
Rhodococcus rhodochrous 172, Rhodococcus sp. 400
-
4 * 45000, SDS-PAGE
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hanging-drop vapour diffusion method in the presence of NaH2PO4 and K2HPO4 as precipitants. X-ray diffraction data are collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120. The asymmetric unit contains two molecules
-
crystal structure of mutant enzyme Y201F, Y385F, and N300D
-
hanging drop vapor diffusion, hanging drops containing 4 mg/ml protein, 100 mM potassium phosphate, pH 7.0, 0.05 mM glutathione, 30 mM sodium sulfite, 0.02 mM FAD, 450 mM ammonium sulfate are equilibrated at 30C for 7-10 days against a well solution of similar composition, but containing 900 mM ammonium sulfate, crystals of R220Q PHBH diffract to approx. 2.0 A
-
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
-
enzyme bound in a crystal is able to convert 4-hydroxybenzoate
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
6.5
-
optimal stability
6.4
8
-
at 0C or at 25C, 50 h, stable
7
7.5
-
optimal stability
9
9.5
-
optimal stability
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4
-
-
24 h, 40% loss of activity without stabilizer, 10% loss of activity in presence of 4-hydroxybenzoate, FAD and EDTA
4
-
-
50 mM phosphate buffer, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 10 mM EDTA, loss of activity after 72 h
21
-
-
room temperature, without glycerol and EDTA, complete inactivation after 20 h
25
-
-
50 h, stable; pH 6.4-8.0, 50 h, stable
25
-
-
pH 5.5-8.5, stable
40
-
-
pH 5.5-7.5, stable
40
-
-
pH 8.0, stable
50
-
-
50% loss of activity after 15 min, strain 420; 50% loss of activity after 45 min, strain 557; 50% loss of activity after 9 min, strain IG
50
-
-
50% loss of activity after 180 min
50
-
-
50% loss of activity after 50 min
50
-
-
50% loss of activity after 37 min
50
-
-
10 min, 60% loss of activity, in presence of 0.1 mM 4-hydroxybenzoate the enzyme retains 80% of its activity
52
-
-
pH 7.5, 20 mM potassium phosphate buffer, 20 min, stable
60
-
-
pH 5.5-6.5, rather stable
60
-
-
in presence of 1 mM 4-hydroxybenzoate, 40% loss of activity after 20 min
65
-
-
1 mM 4-hydroxybenzoate, 95% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
stable to freezing and thawing
-
very stable, even in absence of stabilizing agents
-
extremely unstable, undergoing rapid inactivation unless protected by substrates and other stabilizing agents
-
enzyme rapidly loses activity in dilute solutions, below 2 mg/ml, without stabilizer
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4C, 24 h, 40% loss of activity without stabilizer, 10% loss of activity in presence of 4-hydroxybenzoate, FAD and EDTA
-
4C, 50 mM phosphate buffer, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 10 mM EDTA, loss of activity after 72 h
-
-70C, 5 mM potassium phosphate, pH 7.5, 5% glycerol, stable for several months
-
0C-4C, as a precipitate under a solution of 50 mM potassium phosphate and 0.5 mM EDTA, pH 6.5-7.0, with 70% saturated ammonium sulfate, idefinitely stable
-
4C, as ammonium sulfate paste, indefinitely stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme
-
recombinant PHBH
-
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
-
recombinat enzyme, cloned in Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
overexpression in Escherichia coli
-
When expressed in Escherichia coli, p-hydroxybenzoate hydroxylase transforms p-hydroxybenzoate into protocatechuate
-
mobA gene from pSEB2 cloned into pZR80. Random mutations introduced into the mobA gene and cloned into pCR2.1-TOPO and transformed into Escherichia coli Top10
-, Q6SSJ6
expression in Escherichia coli
-
expression in Escherichia coli, mutant enzyme S212A
-
expression of mutant enzymes Y201F and Y385F in Escherichia coli
-
expression of wild-type and E49Q mutant PHPH in Escherichia coli
-
mutant enzymes K297M, N300D and Y385F
-
plasmid mutagenesis for high-level expression of 4-hydroxybenzoate hydroxylase
-
mutant enzymes R42S and R42K expressed in transformed Escherichia coli TG2 cells
-
expressed in Escherichia coli
Q9R9T1
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and benzoate drastically decreases the transcription of this gene. Repression is mediated by pobR, the transcriptional activator of pobA gene
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
A16T/S394P/D416A
-, Q6SSJ6
low ability to hydroxylate 3-aminophenol
A400G
-, Q6SSJ6
transforms 3-aminophenol with efficiency almost like mutant A400G/K429R
A400G/K429R
-, Q6SSJ6
among mutants, highest enzymatic activity to hydroxylate 3-aminophenol
H135P
-, Q6SSJ6
alters the enzyme's substrate specificity; low ability to hydroxylate 3-aminophenol
H135P/I217L/Y304H
-, Q6SSJ6
low ability to hydroxylate 3-aminophenol
K326I
-, Q6SSJ6
lacks the ability to transform phenol to catechol as the wild-type
K429R
-, Q6SSJ6
can not transform 3-aminophenol at all
N102T/I259S/V399M
-, Q6SSJ6
low ability to hydroxylate 3-aminophenol
N227H
-, Q6SSJ6
is not able to transform 3-aminophenol
N227H/D416A
-, Q6SSJ6
almost has the same transformation efficiency as mutant N227H/Q292R/D416A
N227H/Q292R/D416A
-, Q6SSJ6
among mutants, highest enzymatic activity to hydroxylate 3-aminophenol
Q292R
-, Q6SSJ6
is not able to transform 3-aminophenol
R152L/F364V
-, Q6SSJ6
low ability to hydroxylate 3-aminophenol
V257A
-, Q6SSJ6
mutation enables the mutant to transform phenol to catechol, also has enhanced ability to transform resorcinol, hydroquinone, p-hydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3-chlorophenol, 4-chlorophenol, 4-chlororesorcinol, and 4-nitrophenol, thus broadens the substrate range. Is not capable of hydroxylating benzoate, o-hydroxybenzoate (salicylate), 2,4-dihydroxybenzoate, 2,6-dihydroxybenzoate, 2-chlorophenol, 3-aminophenol, 4-methoxybenzoate, 3-toluate, o-cresol, m-cresol, or p-cresol as the wild-type
A400G
Comamonas testosteroni GZ39
-
transforms 3-aminophenol with efficiency almost like mutant A400G/K429R
-
H135P
Comamonas testosteroni GZ39
-
alters the enzyme's substrate specificity; low ability to hydroxylate 3-aminophenol
-
K326I
Comamonas testosteroni GZ39
-
lacks the ability to transform phenol to catechol as the wild-type
-
V257A
Comamonas testosteroni GZ39
-
mutation enables the mutant to transform phenol to catechol, also has enhanced ability to transform resorcinol, hydroquinone, p-hydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3-chlorophenol, 4-chlorophenol, 4-chlororesorcinol, and 4-nitrophenol, thus broadens the substrate range. Is not capable of hydroxylating benzoate, o-hydroxybenzoate (salicylate), 2,4-dihydroxybenzoate, 2,6-dihydroxybenzoate, 2-chlorophenol, 3-aminophenol, 4-methoxybenzoate, 3-toluate, o-cresol, m-cresol, or p-cresol as the wild-type
-
D38A
-
kcat/KM for 4-hydroxybenzoate is 16.8fold higher than wild-type value
D38Y
-
kcat/KM for 4-hydroxybenzoate is 11.8fold higher than wild-type value
D38Y/T42R
-
kcat/KM for 4-hydroxybenzoate is 32fold higher than wild-type value
T42R
-
kcat/KM for 4-hydroxybenzoate is 7.2fold higher than wild-type value
E49Q
-
investigation of oxygen half-reaction; mutation enhances the positive charge in the active site of PHBH, rate of hydroxylation is above that of wild-type, the rate of release of product is slower than the rate of return of the flavin to the oxidized state
E49Q
-
mutant has lost the ability in the oxidized state to rapidly exchange the product, i.e., 3,4-dihydroxybenzoate, for the substrate, p-hydroxybenzoate
H72N
-
investigation of oxygen half-reaction; rate of turnover is only about 8% of wild-type enzyme at all pH values
H72N
-
disruption of proton-transfer network, kinetic analysis
K297M
-
decreased positive charge in active site, about 35fold slower hydroxylation rate than the wild-type enzyme. Substitution of 8-Cl-FAD in the mutant gives about 1.8fold increase in hydroxylation rate compared to the wild-type enzyme
K297M
-
investigation of oxygen half-reaction; mutation decreases the positive charge in the active site of PHBH but does not interfere with with the H-bond network, 25fold decrease in the rate of hydroxylation compared to wild-type enzyme
N300D
-
mutation has profound effect on enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interaction of the carboxamide group with the flavin O(2) atom, and the alpha-helix H10 that begins at residue 297 is displaced, altering its dipole interaction with the flavin ring
N300D
-
330fold reduced reduction rate of the flavin of the enzyme by NADPH compared to wild-type enzyme, redox potential of the flavin is 20-40mV lower than that of the wild-type enzyme. The mutation interferes with the orientation of pyridine nucleotide and flavin during reduction, stabilizes flavin C(4a) intermediates, prevents substrate ionization, and alters the rates and strengths of ligand binding
N300D
-
decreased positive charge in active site, about 35fold slower hydroxylation rate than the wild-type enzyme, Substitution of 8-Cl-FAD in the mutant gives about 1.8fold increase in hydroxylation rate compared to the wild-type enzyme
P293S
-
mutation decreases the stability of the folded mutant protein compared to the wild-type PHBH
R220Q
-
1% of wild-type activity, lower affinity to 4-hydroxybenzoate than wild-type
S212A
-
the turnover of the substrate 2,4-dihydroxybenzoate is 1.5-fold faster than the rate observed with the wild-type
Y201F
-
crystals differ from the wild-type enzyme at two surface positions, 228 and 249
Y201F
-
less than 6% of the activity of the wild-type enzyme. Reduction of FAD by NADPH is slower by 10fold, when the mutant enzyme-4-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation
Y201F
-
investigation of oxygen half-reaction
Y385F
-
crystals differ from the wild-type enzyme at two surface positions, 228 and 249
Y385F
-
less than 6% of the activity of the wild-type enzyme. Reduction of FAD by NADPH is slower by 100fold, the mutant enzyme reacts with oxygen to form 25% oxidized enzyme and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. The mutant also hydroxylates the product 3,4-dihydroxybenzoate to form gallic acid
Y385F
-
mutant enzyme with a disrupted hydrogen-bonding network, substitution of 8-Cl-FAD in the mutant gives about 1.5fold increase in hydroxylation rate compared to the wild-type enzyme
Y385F
-
in the oxygen half-reaction, the rate of hydroxylation is 25fold slower than that for the wild-type enzyme at pH 6.5, in contrast to wild-type enzyme there is some formation of H2O2 in the reaction; investigation of oxygen half-reaction
H162D
-
no reliable turnover rate due to impaired NADPH binding
H162K
-
less efficient than wild-type enzyme due to a clear increase in the apparent Km-value for NADPH
H162N
-
no reliable turnover rate due to impaired NADPH binding
H162R
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
H162S
-
no reliable turnover rate due to impaired NADPH binding
H162T
-
no reliable turnover rate due to impaired NADPH binding
H162Y
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
R269D
-
no reliable turnover rate due to impaired NADPH binding
R269K
-
rather efficient enzyme with similar catalytic properties as wild-type enzyme
R269N
-
no reliable turnover rate due to impaired NADPH binding
R269S
-
less efficient than wild-type enzyme due to a clear increase in the apparent Km-value for NADPH
R269T
-
no reliable turnover rate due to impaired NADPH binding
R269Y
-
no reliable turnover rate due to impaired NADPH binding
R42K
-
low activity results from impaired binding of NADPH
R42S
-
low activity results from impaired binding of NADPH
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
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
degradation
-
in soil conditions, Phomopsis liquidambari effectively decomposes 99% of the available 4-hydroxybenzoic acid within 48 h. 4-Hydroxybenzoic acid hydroxylase activity is present in a high level early at 20 h, followed by 3,4-dihydroxybenzoic acid decarboxylase which reaches its highest relative activity at 24 h, and finally catechol 1,2-dioxygenase exhibits peak activity at 32 h
degradation
Phomopsis liquidambaris B3
-
in soil conditions, Phomopsis liquidambari effectively decomposes 99% of the available 4-hydroxybenzoic acid within 48 h. 4-Hydroxybenzoic acid hydroxylase activity is present in a high level early at 20 h, followed by 3,4-dihydroxybenzoic acid decarboxylase which reaches its highest relative activity at 24 h, and finally catechol 1,2-dioxygenase exhibits peak activity at 32 h
-
analysis
-
a bienzyme-based Clark electrode is developed for the interference-free determination of L-glutamate. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (EC 1.4.1.3) in combination with p-hydroxybenzoate hydroxylase (EC 1.14.13.2). The enzymes are entrapped by a poly(carbamoyl) sulfonate hydrogel on a Teflon membrane