Literature summary for 1.14.13.2 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 .

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Crystallization (Commentary)

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

Protein Variants

Protein Variants	Comment	Organism
L199A	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is unaltered compared to the wild-type enzyme	Pseudomonas aeruginosa
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
L199D	site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
L199D/Y385F	site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
L199G	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
L199G/Y385A	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
L199G/Y385F	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
L199H	site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
L199K	site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
L199S	site-directed mutagenesis, the mutant enzyme is almost inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
L199V	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
L199V/Y385A	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
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
L199V/Y385V	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is increased compared to the wild-type enzyme	Pseudomonas aeruginosa
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
Y385A	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly increased compared to the wild-type enzyme	Pseudomonas aeruginosa
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
Y385S	site-directed mutagenesis, the mutant enzyme is inactive with 3,4-dihydroxybenzoate	Pseudomonas aeruginosa
Y385T	site-directed mutagenesis, the activity of the mutant with 3,4-dihydroxybenzoate is slightly reduced compared to the wild-type enzyme	Pseudomonas aeruginosa
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]

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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	-	-

Reaction

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	-	?
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	-	?
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	-	?
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	-	?
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	-	?
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	-	?
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	-	?
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	-	?
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	-	?
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	?	-	-
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	?	-	-
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	?	-	-
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	?	-	-
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	?	-	-
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	?	-	-
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	?	-	-
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

Synonyms	Comment	Organism
p-hydroxybenzoate hydroxylase	-	Pseudomonas aeruginosa
PHBH	-	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
0.33	-	3,4-dihydroxybenzoate	wild-type enzyme, pH 7.5, 30°C	Pseudomonas aeruginosa
0.45	-	4-hydroxybenzoate	mutant L199V/Y385F, pH 7.5, 30°C	Pseudomonas aeruginosa
4.4	-	3,4-dihydroxybenzoate	mutant L199V/Y385F, pH 7.5, 30°C	Pseudomonas aeruginosa
8.9	-	4-hydroxybenzoate	wild-type enzyme, pH 7.5, 30°C	Pseudomonas aeruginosa

pH Optimum

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

Cofactor

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

General Information

General Information	Comment	Organism
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]

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