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Literature summary extracted from

  • Bernhardt, F.H.; Bill, E.; Trautwein, A.X.; Twilfer, H.
    4-Methoxybenzoate monooxygenase from Pseudomonas putida: isolation, biochemical properties, substrate specificity, and reaction mechanisms of the enzyme components (1988), Methods Enzymol., 161, 281-294.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.14.99.15 Putidamonooxin essential part of the enzyme system, O2-activating 2Fe-2S-protein, identified by EPR and Mössbauer spectroscopy Pseudomonas putida

General Stability

EC Number General Stability Organism
1.14.99.15 4-methoxybenzoate, stabilizes Pseudomonas putida
1.14.99.15 ethanol, 5-15% v/v, stabilizes activity in buffer and crude extract Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.99.15 0.00063
-
NADH reconstituted enzyme Pseudomonas putida
1.14.99.15 0.03
-
Putidamonooxin
-
Pseudomonas putida
1.14.99.15 0.14
-
NADPH reconstituted enzyme Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.99.15 Fe2+ mononuclear non-heme iron protein, 2Fe-2S cluster Pseudomonas putida
1.14.99.15 Fe2+ both enzyme components are iron-sulfur proteins Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.99.15 41500
-
3-4 * 41500, putidamonooxin, SDS-PAGE Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.99.15 4-methoxybenzoate + NADH + O2 Pseudomonas putida
-
4-hydroxybenzoate + NAD+ + H2O + formaldehyde
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.14.99.15 Pseudomonas putida
-
-
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.14.99.15 aerobic conditions, 0-4°C, 50% activity lost within 24 h Pseudomonas putida

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.99.15
-
Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.99.15 4-methoxybenzoate + reduced acceptor + O2 = 4-hydroxybenzoate + formaldehyde + acceptor + H2O the bacterial enzyme is a two-component enzyme, consisting of an iron-sulfur flavoprotein (FMN), NADH-putidamonooxin-reductase and a ferredoxin-type, oxygen-activating protein, putidamonooxin Pseudomonas putida

Storage Stability

EC Number Storage Stability Organism
1.14.99.15 -20°C, freeze-dried partially purified reductase stable for months without loss of activity, purified reductase is 3-4 weeks stable after addition of NADH in N2-atmosphere Pseudomonas putida
1.14.99.15 0-4°C, more than 24 days stable in crude extract under anaerobic conditions Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.99.15 3,4-dimethoxybenzoate + NADH + O2
-
Pseudomonas putida 4-hydroxy-3-methoxybenzoate + NAD+ + H2O + formaldehyde
-
?
1.14.99.15 3,4-methylenedioxybenzoate + NADH + O2 piperonylate Pseudomonas putida 3,4-dihydroxybenzoate + NAD+ + H2O + formaldehyde protocatechuate ?
1.14.99.15 3-hydroxybenzoate + NADH + O2 partial uncoupler Pseudomonas putida 3,4-dihydroxybenzoate + NAD+ + H2O in the uncoupled part of the reaction, 3-hydroxybenzoate is not hydroxylated and H2O2 is a product, too ?
1.14.99.15 3-methoxybenzoate + NADH + O2 partial uncoupler Pseudomonas putida 3-hydroxybenzoate + NAD+ + H2O + formaldehyde in the uncoupled part of the reaction 3-methoxybenzoate is not hydroxylated, and H2O2 is a product, too ?
1.14.99.15 3-nitro-4-methoxybenzoate + NADH + O2
-
Pseudomonas putida 3-nitro-4-hydroxybenzoate + NAD+ + H2O + formaldehyde
-
?
1.14.99.15 3-phenyl-4-methoxybenzoate + NADH + O2 partial uncoupler Pseudomonas putida 3-phenyl-4-hydroxybenzoate + NAD+ + H2O + formaldehyde in the uncoupled part of the reaction 3-phenyl-4-methoxybenzoate is not hydroxylated and H2O2 is also a product of the reaction ?
1.14.99.15 4-aminobenzoate + NADH + O2
-
Pseudomonas putida 4-amino-3-hydroxybenzoate + NAD+ + H2O
-
?
1.14.99.15 4-ethoxybenzoate + NADH + O2
-
Pseudomonas putida 4-hydroxybenzoate + acetaldehyde + NAD+ + H2O
-
?
1.14.99.15 4-Hydroxy-3-methoxybenzoate + NADH + O2 vanillate, partial uncoupler Pseudomonas putida ?
-
?
1.14.99.15 4-hydroxybenzoate + NADH + O2
-
Pseudomonas putida 3,4-dihydroxybenzoate + NAD+ + H2O
-
?
1.14.99.15 4-methoxybenzoate + NADH + O2
-
Pseudomonas putida 4-hydroxybenzoate + NAD+ + H2O + formaldehyde
-
?
1.14.99.15 4-methoxybenzoate + NADPH + O2
-
Pseudomonas putida 4-hydroxybenzoate + NADP+ + H2O + formaldehyde
-
r
1.14.99.15 4-methylbenzoate + NADH + O2 p-toluate, partial uncoupler Pseudomonas putida 4-carboxybenzylalcohol + NAD+ + H2O in the uncoupled part of the reaction, p-toluate is not hydroxylated and H2O2 is a product, too ?
1.14.99.15 4-methylmercaptobenzoate + NADH + O2
-
Pseudomonas putida ?
-
?
1.14.99.15 4-vinylbenzoate + NADH + O2 external dioxygenase reaction by substrate induced modulation Pseudomonas putida 4-glycylbenzoate + NAD+ + H2O
-
?
1.14.99.15 additional information overview: substrates that are not oxygenized while NADH-oxidation and O2-consumption are catalyzed, such as: benzoate, 3-chlorobenzoate, 4-chlorobenzoate, 2-hydroxybenzoate, 4-bromobenzoate, 2-aminobenzoate, 3-aminobenzoate, 4-trifluoromethylbenzoate, 4-tert-butylbenzoate Pseudomonas putida additional information uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction ?
1.14.99.15 additional information overview: substrates being absolutely planar aromatic rings with a directly bound dissociable carboxy group are oxygenated under stoichiometric consumption of O2 and NADH Pseudomonas putida additional information uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction ?
1.14.99.15 N,N'-dimethyl-4-aminobenzoate + NADH + O2
-
Pseudomonas putida 4-aminobenzoate + NAD+ + H2O + formaldehyde
-
?
1.14.99.15 N-methyl-4-aminobenzoate + NADH + O2
-
Pseudomonas putida 4-aminobenzoate + NAD+ + H2O + formaldehyde
-
?

Subunits

EC Number Subunits Comment Organism
1.14.99.15 oligomer 3-4 * 41500, putidamonooxin, SDS-PAGE Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.99.15 30
-
assay at Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.99.15 8
-
-
Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.99.15 FMN
-
Pseudomonas putida
1.14.99.15 NADH requirement, can be replaced by NADPH with 40% efficiency Pseudomonas putida
1.14.99.15 NADPH can replace NADH with 40% efficiency Pseudomonas putida