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Information on EC 1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase and Organism(s) Pseudomonas aeruginosa and UniProt Accession P20586
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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 aeruginosa
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, 4-hba 3-monooxygenase, 4-hba 3-hydroxylase, phbad, 4hba 3-hydroxylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
4-HBA-3-hydroxylase
-
-
-
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4-hydroxybenzoate 3-hydroxylase
-
-
-
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4-hydroxybenzoate 3-monooxygenase
-
-
-
-
4-hydroxybenzoate monooxygenase
-
-
-
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4-hydroxybenzoic hydroxylase
-
-
-
-
oxygenase, 4-hydroxybenzoate 3-mono-
-
-
-
-
p-hydroxybenzoate hydroxylase
p-hydroxybenzoate-3-hydroxylase
-
-
-
-
p-hydroxybenzoic acid hydrolase
-
-
-
-
p-hydroxybenzoic acid hydroxylase
-
-
-
-
p-hydroxybenzoic hydroxylase
-
-
-
-
para-hydroxybenzoate hydroxylase
-
-
-
-
PHBAD
-
-
-
-
PHBH
PHBHase
-
-
-
-
POHBase
-
-
-
-
p-hydroxybenzoate hydroxylase
-
-
-
-
PHBH
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-hydroxybenzoate + NADPH + H+ + O2 = 3,4-dihydroxybenzoate + NADP+ + H2O
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 = 3,4-dihydroxybenzoate + NADP+ + H2O
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 = 3,4-dihydroxybenzoate + NADP+ + H2O
catalytic cycle of enzyme PHBH, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
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
-
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
2,4-dihydroxybenzoate + NADPH + O2
2,3,4-trihydroxybenzoate + 2,4,5-trihydroxybenzoate + NADP+ + H2O
-
-
?
4-hydroxybenzoate + NADPH + H+ + O2
3,4-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
?
3,4-dihydroxybenzoic acid + NADPH + H+ + O2
gallic acid + NADP+ + H2O
-
weak activity
-
-
?
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
3,4-dihydroxybenzoate + NADPH + H+ + O2
gallic acid + NADP+ + H2O
activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme
-
-
?
3,4-dihydroxybenzoate + NADPH + H+ + O2
gallic acid + NADP+ + H2O
good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA
-
-
?
additional information
?
-
3,4-dihydroxybenzoate is no substrate for hydroxylation by the enzyme. But PHBH can bind to other benzoate derivatives in addition to 4-hydroxybenzoate
-
-
-
additional information
?
-
-
mutant enzyme Y385F hydroxylates 3,4-dihydroxybenzoate to form gallic acid
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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-hydroxybenzoate + NADPH + H+ + O2
3,4-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
?
4-hydroxybenzoate + NADPH + H+ + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
4-hydroxybenzoate + NADPH + O2
protocatechuate + NADP+ + H2O
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
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
FAD
-
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
FAD
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thermodynamic and kinetic constants of the enzyme reconstituted with 8-substituted flavins
INHIBITOR
ORGANISM
UNIPROT
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
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.048
3,4-dihydroxybenzoate
pH 8.0, 30°C, mutant DA015, with NADPH
0.12
3,4-dihydroxybenzoate
pH 8.0, 30°C, mutant DA016, with NADPH
0.048
4-hydroxybenzoate
pH 8.0, 30°C, wild-type enzyme, with NADPH
0.15702
3,4-dihydroxybenzoic acid
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mutant Y385F/T294A, at pH 8.0 and 30°C
additional information
additional information
apparent kinetic parameters are estimated through non-linear regression of the Michaelis-Menten equation
-
additional information
additional information
-
apparent kinetic parameters are estimated through non-linear regression of the Michaelis-Menten equation
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4.1
3,4-dihydroxybenzoate
pH 8.0, 30°C, mutant DA016, with NADPH
7.6
4-hydroxybenzoate
pH 8.0, 30°C, wild-type enzyme, with NADPH
1.69
3,4-dihydroxybenzoic acid
-
mutant Y385F/T294A, at pH 8.0 and 30°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
34.2
3,4-dihydroxybenzoate
pH 8.0, 30°C, mutant DA016, with NADPH
62.5
3,4-dihydroxybenzoate
pH 8.0, 30°C, mutant DA015, with NADPH
158.3
4-hydroxybenzoate
pH 8.0, 30°C, wild-type enzyme, with NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.26
-
mutant enzyme Y385F, with 4-hydroxybenzoate as substrate, at pH 8.0 and 30°C
0.45
-
mutant enzyme Y385F, with 3,4-dihydroxybenzoic acid as substrate, at pH 8.0 and 30°C
1.04
-
mutant enzyme Y385F/T294A, with 4-hydroxybenzoate as substrate, at pH 8.0 and 30°C
1.86
-
mutant enzyme Y385F/T294A, with 3,4-dihydroxybenzoic acid as substrate, at pH 8.0 and 30°C
16.85
-
wild type enzyme, with 4-hydroxybenzoate as substrate, at pH 8.0 and 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. The L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wasts NADPH by producing H2O2, molecular mechanism underlying this higher catalytic activity, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, overview
metabolism
the NADPH-dependent 4-HBA hydroxylase from Pseudomonas aeruginosa (Pa PobA) natively catalyzes the first step in gallic acid biosynthesis
additional information
additional information
in wild-type PobA structure (PDB ID 1IUW), the productive binding mode is described by the hydrogen-bonding networks stabilizing 4-HBA and positioning the 3-carbon toward FAD for hydroxylation, comparison with enzyme mutants Da015 and DA016
additional information
-
in wild-type PobA structure (PDB ID 1IUW), the productive binding mode is described by the hydrogen-bonding networks stabilizing 4-HBA and positioning the 3-carbon toward FAD for hydroxylation, comparison with enzyme mutants Da015 and DA016
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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 30°C 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
purifed enzyme mutant Y385F in complex with 3,4-dihydroxybenzoate, X-ray diffraction structure determination and analysis
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
L199A
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
L199A/Y385F
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme
L199D
site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
L199D/Y385F
site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
L199G
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
L199G/Y385A
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
L199G/Y385F
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
L199H
site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate
L199K
site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate
L199R/T294C/Y385M
site-directed and random mutagenesis, mutant DA015, in the DA015 model, L199R supports Y201 and forms a new contact to the ligand 3-hydroxyl. Y385M makes space, no substitution occurs at V47, which maintains close hydrophobic packing against L199R, and T294C loosens the helix for increased flexibility and improved backbone hydrogen bonding to the 4-hydroxyl. The mutation orients 3,4-DHBA such that the 5-carbon is optimally exposed to FAD for hydroxylation
L199S
site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate
L199V
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
L199V/Y385A
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
L199V/Y385F
site-directed mutagenesis, the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-dihydroxybenzoate, which is necessary for initiating hydroxylation, and the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB
L199V/Y385V
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme
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
V47I/L199N/T294A/Y385I
site-directed and random mutagenesis, mutant DA016, in the DA016 model, L199N forms interactions stabilizing S212 and to the ligand 3-hydroxyl. Y385I creates space, V47I braces L199N to minimize side-chain mobility, and T294A allows P293 to move closer to 3,4-DHBA. The mutation orients 3,4-DHBA such that the 5-carbon is optimally exposed to FAD for hydroxylation
Y385A
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme
Y385F
site-directed mutagenesis, the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-dihydroxybenzoate, which is necessary for initiating hydroxylation
Y385S
site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
Y385T
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly reduced compared to the wild-type enzyme
Y385V
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly reduced compared to the wild-type enzyme
E49Q
H72N
K297M
N300D
P293S
-
mutation decreases the stability of the folded mutant protein compared to the wild-type PHBH
Y201F
Y385F
Y385F/T294A
-
the mutant displays much higher activity toward 3,4-dihydroxybenzoic acid than the wild type enzyme
additional information
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
E49Q
-
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
H72N
-
rate of turnover is only about 8% of wild-type enzyme at all pH values
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
-
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
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
Y385F
-
crystals differ from the wild-type enzyme at two surface positions, 228 and 249
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
-
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
-
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
Y385F
-
the mutant displays higher activity toward 3,4-dihydroxybenzoic acid than the wild type enzyme
additional information
for directed evolution of an NADPH-dependent monooxygenase Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to engineer high activity for a non-native substrate, 3,4-dihydroxybenzoic acid (3,4-DHBA) which is the precursor of a natural product antioxidant, gallic acid (GA), an aerobic, growth-based selection platform is founded on NADP(H) redox balance restoration in Escherichia coli strain MX203. Application in the high-throughput evolution of the oxygenase. A single round of selection followed by a facile growth assay enables Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Reorganization of active site hydrogen bond network. Mutant variants with roughly 8fold improved apparent catalytic efficiency (kcat/KM) for 3,4-DHBA, compared to the wild type, are obtained. Engineered Escherichia coli strain (MX203) has a growth deficiency linked to NADPH/NADP+ imbalance. The perturbed redox state results from deletion of central metabolism genes, glucose-6-phosphate isomerase gene pgi and phosphogluconate dehydratase gene edd, a critical rebalancing tool, soluble pyridine nucleotide transhydrogenase gene udhA, and a significant sink for reduced nicotinamide cofactors, NAD(P)H:quinone oxidoreductase gene qor. These disruptions cause MX203 to exhibit poor growth in glucose minimal media. Possibility of the universal application of this selection platform in engineering NADPH-dependent oxidoreductases. Mutants modeling and docking of 3,4-DHBA, overview
additional information
-
for directed evolution of an NADPH-dependent monooxygenase Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to engineer high activity for a non-native substrate, 3,4-dihydroxybenzoic acid (3,4-DHBA) which is the precursor of a natural product antioxidant, gallic acid (GA), an aerobic, growth-based selection platform is founded on NADP(H) redox balance restoration in Escherichia coli strain MX203. Application in the high-throughput evolution of the oxygenase. A single round of selection followed by a facile growth assay enables Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Reorganization of active site hydrogen bond network. Mutant variants with roughly 8fold improved apparent catalytic efficiency (kcat/KM) for 3,4-DHBA, compared to the wild type, are obtained. Engineered Escherichia coli strain (MX203) has a growth deficiency linked to NADPH/NADP+ imbalance. The perturbed redox state results from deletion of central metabolism genes, glucose-6-phosphate isomerase gene pgi and phosphogluconate dehydratase gene edd, a critical rebalancing tool, soluble pyridine nucleotide transhydrogenase gene udhA, and a significant sink for reduced nicotinamide cofactors, NAD(P)H:quinone oxidoreductase gene qor. These disruptions cause MX203 to exhibit poor growth in glucose minimal media. Possibility of the universal application of this selection platform in engineering NADPH-dependent oxidoreductases. Mutants modeling and docking of 3,4-DHBA, overview
additional information
molecular mechanism underlying this higher catalytic activity of some enzyme mutants, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, overview
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0°C-4°C, 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
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene pobA, construction and selection of PobA library, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)
plasmid mutagenesis for high-level expression of 4-hydroxybenzoate hydroxylase
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Entsch, B.
Hydroxybenzoate hydroxylase
Methods Enzymol.
188
138-147
1990
Pseudomonas aeruginosa
Entsch, B.; Ballou, D.P.
Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa [published erratum appears in Biochim Biophys Acta 1990 Mar 29;1038(1):139]
Biochim. Biophys. Acta
999
313-322
1989
Pseudomonas aeruginosa
Moran, G.R.; Entsch, B.; Palfey, B.A.; Ballou, D.P.
Mechanistic insights into p-hydroxybenzoate hydroxylase from studies of the mutant Ser212Ala
Biochemistry
38
6292-6299
1999
Pseudomonas aeruginosa (P20586)
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)
Lah, M.S.; Palfey, B.A.; Schreuder, H.A.; Ludwig, M.L.
Crystal structures of mutant Pseudomonas aeruginosa p-hydroxybenzoate hydroxylases: The Tyr201Phe, Tyr385Phe, and Asn300Asp variants
Biochemistry
33
1555-1564
1994
Pseudomonas aeruginosa
Entsch, B.; Palfey, B.A.; Ballou, D.P.; Massey, V.
Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants
J. Biol. Chem.
266
17341-17349
1991
Pseudomonas aeruginosa
Palfey, B.A.; Entsch, B.; Ballou, D.P.; Massey, V.
Changes in the catalytic properties of p-hydroxybenzoate hydroxylase caused by the mutation Asn300Asp
Biochemistry
33
1545-1554
1994
Pseudomonas aeruginosa
Moran, G.R.; Entsch, B.
Plasmid mutagenesis by PCR for high-level expression of para-hydroxybenzoate hydroxylase
Protein Expr. Purif.
6
164-168
1995
Pseudomonas aeruginosa
Ortiz-Maldonado, M.; Gatti, D.; Ballou, D.P.; Massey, V.
Structure-function correlation of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins
Biochemistry
38
16636-16647
1999
Pseudomonas aeruginosa
Ortiz-Maldonado, M.; Aeschliman, S.M.; Ballou, D.P.; Masey, V.
Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl flavin
Biochemistry
40
8705-8716
2001
Pseudomonas aeruginosa
Palfey, B.A.; Basu, R.; Frederick, K.K.; Entsch, B.; Ballou, D.P.
Role of protein flexibility in the catalytic cycle of p-hydroxybenzoate hydroxylase elucidated by the Pro293Ser mutant
Biochemistry
41
8438-8446
2002
Pseudomonas aeruginosa
Ortiz-Maldonado, M.; Entsch, B.; Ballou, D.P.
Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase
Biochemistry
42
11234-11242
2003
Pseudomonas aeruginosa
Ortiz-Maldonado, M.; Entsch, B.; Ballou, D.P.
Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis
Biochemistry
43
15246-15257
2004
Pseudomonas aeruginosa
Ortiz-Maldonado, M.; Cole, L.J.; Dumas, S.M.; Entsch, B.; Ballou, D.P.
Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover
Biochemistry
43
1569-1579
2004
Pseudomonas aeruginosa
Wang, J.; Ortiz-Maldonado, M.; Entsch, B.; Massey, V.; Ballou, D.; Gatti, D.L.
Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase
Proc. Natl. Acad. Sci. USA
99
608-613
2002
Pseudomonas aeruginosa (P20586)
Frederick, K.K.; Palfey, B.A.
Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase
Biochemistry
44
13304-13314
2005
Pseudomonas aeruginosa (P20586)
Chen, Z.; Shen, X.; Wang, J.; Wang, J.; Yuan, Q.; Yan, Y.
Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway
Biotechnol. Bioeng.
114
2571-2580
2017
Pseudomonas aeruginosa
Maxel, S.; Aspacio, D.; King, E.; Zhang, L.; Acosta, A.P.; Li, H.
A growth-based, high-throughput selection platform enables remodeling of 4-hydroxybenzoate hydroxylase active site
ACS Catal.
10
6969-6974
2020
Pseudomonas aeruginosa (P20586), Pseudomonas aeruginosa, Pseudomonas aeruginosa ATCC 15692 (P20586), Pseudomonas aeruginosa 1C (P20586), Pseudomonas aeruginosa PRS 101 (P20586), Pseudomonas aeruginosa DSM 22644 (P20586), Pseudomonas aeruginosa CIP 104116 (P20586), Pseudomonas aeruginosa LMG 12228 (P20586), Pseudomonas aeruginosa JCM 14847 (P20586)
Moriwaki, Y.; Yato, M.; Terada, T.; Saito, S.; Nukui, N.; Iwasaki, T.; Nishi, T.; Kawaguchi, Y.; Okamoto, K.; Arakawa, T.; Yamada, C.; Fushinobu, S.; Shimizu, K.
Understanding the molecular mechanism underlying the high catalytic activity of p-hydroxybenzoate hydroxylase mutants for producing gallic acid
Biochemistry
58
4543-4558
2019
Pseudomonas aeruginosa (P20586), Pseudomonas aeruginosa ATCC 15692 (P20586), Pseudomonas aeruginosa 1C (P20586), Pseudomonas aeruginosa PRS 101 (P20586), Pseudomonas aeruginosa DSM 22644 (P20586), Pseudomonas aeruginosa CIP 104116 (P20586), Pseudomonas aeruginosa LMG 12228 (P20586), Pseudomonas aeruginosa JCM 14847 (P20586)
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