1.14.13.2: 4-hydroxybenzoate 3-monooxygenase
This is an abbreviated version!
For detailed information about 4-hydroxybenzoate 3-monooxygenase, go to the full flat file.
Word Map on EC 1.14.13.2
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1.14.13.2
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flavin
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fad
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fluorescens
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flavoproteins
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isoalloxazine
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protocatechuate
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3,4-dioxygenase
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massey
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2,4-dihydroxybenzoate
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ballou
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c4a-hydroperoxide
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3-hydroxybenzoate
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gatti
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degradation
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analysis
- 1.14.13.2
- flavin
- fad
- fluorescens
- flavoproteins
- isoalloxazine
- protocatechuate
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3,4-dioxygenase
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massey
- 2,4-dihydroxybenzoate
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ballou
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c4a-hydroperoxide
- 3-hydroxybenzoate
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gatti
- degradation
- analysis
Reaction
Synonyms
4-HBA 3-hydroxylase, 4-HBA 3-monooxygenase, 4-HBA-3-hydroxylase, 4-HBMO, 4-hydroxybenzoate 3-hydroxylase, 4-hydroxybenzoate 3-monooxygenase, 4-hydroxybenzoate hydroxylase, 4-hydroxybenzoate monooxygenase, 4-hydroxybenzoic hydroxylase, 4HBA 3-hydroxylase, An_PhhA, BxeA2040, FAD-dependent 4-hydroxybenzoate hydroxylase, fungal 4-hydroxybenzoate 3-hydroxylase, HBH, m-hydroxybenzoate hydroxylase, MobA, NADPH-dependent 4-HBA hydroxylase, ncgl1032, oxygenase, 4-hydroxybenzoate 3-mono-, p-hydroxybenzoate hydroxylase, p-hydroxybenzoate-3-hydroxylase, p-hydroxybenzoic acid hydrolase, p-hydroxybenzoic acid hydroxylase, p-hydroxybenzoic hydroxylase, PaPobA, para-hydroxybenzoate hydroxylase, PHBAD, PHBH, PHBHase, PHBHCn2, phhA, PobA, pobACg, POHBase, Reut_B4006
ECTree
1.14.13.2 4-hydroxybenzoate 3-monooxygenaseAdvanced search results
results (
results (Engineering
Engineering on EC 1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
A400G
transforms 3-aminophenol with efficiency almost like mutant A400G/K429R
A400G/K429R
among mutants, highest enzymatic activity to hydroxylate 3-aminophenol
H135P
K326I
lacks the ability to transform phenol to catechol as the wild-type
N227H/D416A
almost has the same transformation efficiency as mutant N227H/Q292R/D416A
N227H/Q292R/D416A
among mutants, highest enzymatic activity to hydroxylate 3-aminophenol
V257A
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
-
transforms 3-aminophenol with efficiency almost like mutant A400G/K429R
-
H135P
K326I
-
lacks the ability to transform phenol to catechol as the wild-type
-
V257A
-
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
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D38A
-
kcat/KM for 4-hydroxybenzoate is 16.8fold higher than wild-type value
D38Y
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kcat/KM for 4-hydroxybenzoate is 11.8fold higher than wild-type value
D38Y/T42R
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kcat/KM for 4-hydroxybenzoate is 32fold higher than wild-type value
T42R
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kcat/KM for 4-hydroxybenzoate is 7.2fold higher than wild-type value
E49Q
H72N
K297M
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
N300D
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
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
Y201F
Y385A
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme
Y385F
Y385F/T294A
-
the mutant displays much higher activity toward 3,4-dihydroxybenzoic acid than the wild type enzyme
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
L199A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
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L199D
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site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
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L199R/T294C/Y385M
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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
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V47I/L199N/T294A/Y385I
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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
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Y385A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme
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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
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L199A
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site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
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L199D
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site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
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L199R/T294C/Y385M
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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
-
V47I/L199N/T294A/Y385I
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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
-
L199A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
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L199D
-
site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
-
L199R/T294C/Y385M
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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
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V47I/L199N/T294A/Y385I
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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
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Y385F
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site-directed mutagenesis, the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-dihydroxybenzoate, which is necessary for initiating hydroxylation
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L199A
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site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
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L199D
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site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
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L199R/T294C/Y385M
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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
-
V47I/L199N/T294A/Y385I
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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
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Y385A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme
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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
-
L199A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
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L199D
-
site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate
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L199R/T294C/Y385M
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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
-
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
-
L199A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
-
L199D
-
site-directed mutagenesis, the mutant enzyme is 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
-
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
-
L199A
-
site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme
-
L199D
-
site-directed mutagenesis, the mutant enzyme is 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
-
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
-
H162K
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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
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
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
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
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
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
-
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
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molecular mechanism underlying this higher catalytic activity of some enzyme mutants, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, overview
-
additional information
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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
-
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
-
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
-
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
-
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
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