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Literature summary extracted from

  • 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 (2019), Biochemistry, 58, 4543-4558 .
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.2 purifed enzyme mutant Y385F in complex with 3,4-dihydroxybenzoate, X-ray diffraction structure determination and analysis Pseudomonas aeruginosa

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.2 L199A site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199A/Y385F site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199D site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 L199D/Y385F site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 L199G site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199G/Y385A site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199G/Y385F site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199H site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 L199K site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 L199S site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 L199V site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 L199V/Y385A site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 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 Pseudomonas aeruginosa
1.14.13.2 L199V/Y385V site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 additional information molecular mechanism underlying this higher catalytic activity of some enzyme mutants, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, overview Pseudomonas aeruginosa
1.14.13.2 Y385A site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 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 Pseudomonas aeruginosa
1.14.13.2 Y385S site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate Pseudomonas aeruginosa
1.14.13.2 Y385T site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly reduced compared to the wild-type enzyme Pseudomonas aeruginosa
1.14.13.2 Y385V site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly reduced compared to the wild-type enzyme Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.2 0.022
-
4-hydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 0.039
-
4-hydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 0.042
-
3,4-dihydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 0.052
-
3,4-dihydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa ATCC 15692
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa 1C
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa PRS 101
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa DSM 22644
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa CIP 104116
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa LMG 12228
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas aeruginosa JCM 14847
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?

Organism

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

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 = 3,4-dihydroxybenzoate + NADP+ + H2O catalytic cycle of enzyme PHBH, overview Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa ATCC 15692 gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa 1C gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa PRS 101 gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa DSM 22644 gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa CIP 104116 gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa LMG 12228 gallic acid + NADP+ + H2O
-
?
1.14.13.2 3,4-dihydroxybenzoate + NADPH + H+ + O2 good substrate of enzyme mutants Y385F and L199V/Y385F, poor activity with the wild-type enzyme PobA Pseudomonas aeruginosa JCM 14847 gallic acid + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa ATCC 15692 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa 1C 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa PRS 101 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa DSM 22644 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa CIP 104116 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa LMG 12228 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Pseudomonas aeruginosa JCM 14847 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 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 Pseudomonas aeruginosa ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa ATCC 15692 ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa 1C ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa PRS 101 ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa DSM 22644 ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa CIP 104116 ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa LMG 12228 ?
-
-
1.14.13.2 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 Pseudomonas aeruginosa JCM 14847 ?
-
-

Synonyms

EC Number Synonyms Comment Organism
1.14.13.2 p-hydroxybenzoate hydroxylase
-
Pseudomonas aeruginosa
1.14.13.2 PHBH
-
Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.2 30
-
assay at Pseudomonas aeruginosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.2 0.33
-
3,4-dihydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 0.45
-
4-hydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 4.4
-
3,4-dihydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 8.9
-
4-hydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.2 7.5
-
assay at Pseudomonas aeruginosa

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.2 FAD
-
Pseudomonas aeruginosa
1.14.13.2 NADPH
-
Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
1.14.13.2 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 Pseudomonas aeruginosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.2 7.86
-
3,4-dihydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 20.45
-
4-hydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 84.6
-
3,4-dihydroxybenzoate mutant L199V/Y385F, pH 7.5, 30°C Pseudomonas aeruginosa
1.14.13.2 228.2
-
4-hydroxybenzoate wild-type enzyme, pH 7.5, 30°C Pseudomonas aeruginosa