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Literature summary for 1.14.13.2 extracted from

  • 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 (2020), ACS Catal., 10, 6969-6974 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.14.13.2

Cloned(Commentary)

Cloned (Comment) Organism
gene pobA, construction and selection of PobA library, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) Pseudomonas aeruginosa

Protein Variants

Protein Variants Comment Organism
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 Pseudomonas aeruginosa
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 Pseudomonas aeruginosa
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 Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information apparent kinetic parameters are estimated through non-linear regression of the Michaelis-Menten equation Pseudomonas aeruginosa
0.024
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
0.048
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
0.048
-
 4-hydroxybenzoate pH 8.0, 30°C, wild-type enzyme, with NADPH Pseudomonas aeruginosa
0.12
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa
0.12
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa ATCC 15692
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa 1C
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa PRS 101
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa DSM 22644
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa CIP 104116
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa LMG 12228
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa JCM 14847
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas aeruginosa P20586
-
-
Pseudomonas aeruginosa 1C P20586
-
-
Pseudomonas aeruginosa ATCC 15692 P20586
-
-
Pseudomonas aeruginosa CIP 104116 P20586
-
-
Pseudomonas aeruginosa DSM 22644 P20586
-
-
Pseudomonas aeruginosa JCM 14847 P20586
-
-
Pseudomonas aeruginosa LMG 12228 P20586
-
-
Pseudomonas aeruginosa PRS 101 P20586
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa ATCC 15692 gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa 1C gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa PRS 101 gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa DSM 22644 gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa CIP 104116 gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa LMG 12228 gallic acid + NADP+ + H2O
-
 ?
3,4-dihydroxybenzoate + NADPH + H+ + O2 activity of enzyme mutants DA015 and DA016, no activity with the wild-type enzyme Pseudomonas aeruginosa JCM 14847 gallic acid + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa ATCC 15692 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa 1C 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa PRS 101 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa DSM 22644 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa CIP 104116 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa LMG 12228 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas aeruginosa JCM 14847 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?

Synonyms

Synonyms Comment Organism
4-hydroxybenzoate hydroxylase
-
 Pseudomonas aeruginosa
NADPH-dependent 4-HBA hydroxylase
-
 Pseudomonas aeruginosa
PaPobA
-
 Pseudomonas aeruginosa
PobA
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.9
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
3
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
4
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa
4.1
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa
7.6
-
 4-hydroxybenzoate pH 8.0, 30°C, wild-type enzyme, with NADPH Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Pseudomonas aeruginosa

Cofactor

Cofactor Comment Organism Structure
FAD
-
 Pseudomonas aeruginosa
NADPH
-
 Pseudomonas aeruginosa

General Information

General Information Comment Organism
metabolism the NADPH-dependent 4-HBA hydroxylase from Pseudomonas aeruginosa (Pa PobA) natively catalyzes the first step in gallic acid biosynthesis Pseudomonas aeruginosa
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 Pseudomonas aeruginosa

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
33.3
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa
34.2
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA016, with NADPH Pseudomonas aeruginosa
62.5
-
 3,4-dihydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
79.2
-
 4-hydroxybenzoate pH 8.0, 30°C, mutant DA015, with NADPH Pseudomonas aeruginosa
158.3
-
 4-hydroxybenzoate pH 8.0, 30°C, wild-type enzyme, with NADPH Pseudomonas aeruginosa