Information on EC 1.14.99.15 - 4-methoxybenzoate monooxygenase (O-demethylating)

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The expected taxonomic range for this enzyme is: Proteobacteria

EC NUMBER
COMMENTARY
1.14.99.15
-
RECOMMENDED NAME
GeneOntology No.
4-methoxybenzoate monooxygenase (O-demethylating)
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-methoxybenzoate + AH2 + O2 = 4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
a terminal oxygenase
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4-methoxybenzoate + AH2 + O2 = 4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
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
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dealkylation
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-
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N-demethylation
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-
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O-demethylation
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oxidation
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-
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redox reaction
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reduction
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-
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S-demethylation
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Benzoate degradation
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-
SYSTEMATIC NAME
IUBMB Comments
4-methoxybenzoate,hydrogen-donor:oxygen oxidoreductase (O-demethylating)
The bacterial enzyme consists of a ferredoxin-type protein and an iron-sulfur flavoprotein (FMN). Also acts on 4-ethoxybenzoate, N-methyl-4-aminobenzoate and toluate. The fungal enzyme acts best on veratrate.
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4-methoxybenzoate 4-monooxygenase (O-demethylating)
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-
-
-
4-methoxybenzoate O-demethylase
-
-
-
-
oxygenase, 4-methoxybenzoate 4-mono- (O-demethylating)
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p-anisic O-demethylase
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-
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piperonylate-4-O-demethylase
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-
-
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CAS REGISTRY NUMBER
COMMENTARY
37256-78-3
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
-
the enzyme belongs to the superfamily of heme-dependent cytochrome P450 monooxygenases
physiological function
-
CYP199A2 catalyzes the oxidative demethylation of 4-methoxybenzoic acid and the hydroxylation and desaturation of 4-ethylbenzoic acid, and thus may play a role in lignin degradation by this organism
physiological function
Q2IU02
CYP199A2 is involved in the degradation of ligninolic compounds by the organism
physiological function
-
CYP199A2 catalyzes the oxidative demethylation of 4-methoxybenzoic acid and the hydroxylation and desaturation of 4-ethylbenzoic acid, and thus may play a role in lignin degradation by this organism, CYP199A2 is involved in the degradation of ligninolic compounds by the organism
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evolution
-
the enzyme belongs to the superfamily of heme-dependent cytochrome P450 monooxygenases
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additional information
Q2IU02
substrate-enzyme structure, the enzyme possesses a clearly defined substrate access channel that is formed between the BC loop and the G and G' helices, overview. The 4-methoxybenzoate-bound enzyme has a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate, the 4-methoxybenzoate-bound enzyme has a closed conformation. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate, involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates
additional information
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the 4-methoxybenzoate-bound enzyme has a closed conformation. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate, involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates
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additional information
-
substrate-enzyme structure, the enzyme possesses a clearly defined substrate access channel that is formed between the BC loop and the G and G' helices, overview. The 4-methoxybenzoate-bound enzyme has a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate
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SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-naphthoic acid + AH2 + O2
5-hydroxy-2-naphthoic acid + A + H2O
show the reaction diagram
Q6N8N2
only the wild-type enzyme hydroxylates 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibit a preference for C-5 hydroxylation
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?
2-naphthoic acid + AH2 + O2
7-hydroxy-2-naphthoic acid + A + H2O
show the reaction diagram
Q6N8N2
only the wild-type enzyme hydroxylates 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibit a preference for C-5 hydroxylation
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-
?
2-naphthoic acid + AH2 + O2
8-hydroxy-2-naphthoic acid + A + H2O
show the reaction diagram
Q6N8N2
only the wild-type enzyme hydroxylates 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibit a preference for C-5 hydroxylation
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?
3,4-dimethoxybenzoate + NADH + O2
4-hydroxy-3-methoxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
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?
3,4-methylenedioxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
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piperonylate
protocatechuate
?
3,4-methylenedioxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
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piperonylate
protocatechuate
?
3-chlorobenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
3-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
partial uncoupler
in the uncoupled part of the reaction, 3-hydroxybenzoate is not hydroxylated and H2O2 is a product, too
?
3-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
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partial uncoupler
in the uncoupled part of the reaction, 3-hydroxybenzoate is not hydroxylated and H2O2 is a product, too
?
3-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
partial uncoupler
in the uncoupled part of the reaction, 3-hydroxybenzoate is not hydroxylated and H2O2 is a product, too
?
3-methoxybenzoate + NADH + O2
3-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
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partial uncoupler
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?
3-methoxybenzoate + NADH + O2
3-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
partial uncoupler
in the uncoupled part of the reaction 3-methoxybenzoate is not hydroxylated, and H2O2 is a product, too
?
3-nitro-4-methoxybenzoate + NADH + O2
3-nitro-4-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
3-phenyl-4-methoxybenzoate + NADH + O2
3-phenyl-4-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
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?
3-phenyl-4-methoxybenzoate + NADH + O2
3-phenyl-4-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
partial uncoupler
in the uncoupled part of the reaction 3-phenyl-4-methoxybenzoate is not hydroxylated and H2O2 is also a product of the reaction
?
4-aminobenzoate + NADH + O2
4-amino-3-hydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-chlorobenzaldehyde + NADH + O2
4-chlorobenzoate + NAD+ + H2O
show the reaction diagram
-
-
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?
4-chlorobenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-coumaric acid + AH2 + O2
caffeic acid + A + H2O
show the reaction diagram
Q6N8N2
the F185L mutant exhibits 5.5times higher hydroxylation activity for 4-coumaric acid than the wild-type enzyme, good substrate of enzyme mutant F185L, low activity with enzyme mutant sF185V, F185I, F185G, and F185A, moderate activity with the wild-type enzyme and mutants F185Y, F185W, F185S, and F185T with 4-coumaric acid
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?
4-ethoxybenzoate + NADH + O2
4-hydroxybenzoate + acetaldehyde + NAD+ + H2O
show the reaction diagram
-
-
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?
4-ethylbenzoate + AH2 + O2
4-(1-hydroxyethyl)-benzoate + 4-vinylbenzoate + A + H2O
show the reaction diagram
-
a C-C bond dehydrogenation of an unbranched alkyl group, computational docking of 4-ethylbenzoate into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the beta-methyl group is located over the heme iron by Phe185, this binding orientation is consistent with the observed product profile of exclusive attack at the para substituent, overview
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?
4-ethylbenzoate + AH2 + O2
?
show the reaction diagram
Q2IU02
computational docking study
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-
?
4-ethylbenzoate + AH2 + O2
?
show the reaction diagram
Q6N8N2
computational docking study
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?
4-ethylbenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
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?
4-Hydroxy-3-methoxybenzoate + NADH + O2
?
show the reaction diagram
-
vanillate, partial uncoupler
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?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
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?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
ring hydroxylation
?
4-isopropylbenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
Q2IU02
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
Q6N8N2
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-
-
?
4-methoxybenzoate + NADH + O2
4-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + reduced ferredoxin + O2
4-hydroxybenzoate + formaldehyde + ferredoxin + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
show the reaction diagram
Q2IU02
-
-
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
show the reaction diagram
-
-
-
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?
4-methoxybenzoate + reduced palustrisredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
show the reaction diagram
Q6N8N2
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-
-
?
4-methoxybenzoate + reduced putidaredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized putidaredoxin + H2O
show the reaction diagram
-
very low activity with putidaredoxin
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-
?
4-methoxybenzoic acid + NADH + O2
4-hydroxybenzoate + formaldehyde + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-methylbenzoate + NADH + O2
4-carboxybenzylalcohol + NAD+ + H2O
show the reaction diagram
-
-
-
-
-
4-methylbenzoate + NADH + O2
4-carboxybenzylalcohol + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-methylbenzoate + NADH + O2
4-carboxybenzylalcohol + NAD+ + H2O
show the reaction diagram
-
p-toluate, partial uncoupler
in the uncoupled part of the reaction, p-toluate is not hydroxylated and H2O2 is a product, too
?
4-methylbenzoate + NADH + O2
4-carboxybenzylalcohol + NAD+ + H2O
show the reaction diagram
-
p-toluate, partial uncoupler
in the uncoupled part of the reaction, p-toluate is not hydroxylated and H2O2 is a product, too
?
4-methylbenzoate + NADH + O2
4-carboxybenzylalcohol + NAD+ + H2O
show the reaction diagram
-
p-toluate, partial uncoupler
in the uncoupled part of the reaction, p-toluate is not hydroxylated and H2O2 is a product, too
?
4-methylbenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-methylmercaptobenzoate + NADH + O2
?
show the reaction diagram
-
-
-
-
?
4-methylsalicylic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-t-butylbenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-trifluoromethylbenzoate + NADH + O2
?
show the reaction diagram
-
-
-
-
?
4-vinylbenzoate + NADH + O2
4-glycylbenzoate + NAD+ + H2O
show the reaction diagram
-
external dioxygenase reaction by substrate induced modulation
-
-
?
alkylbenzoates
?
show the reaction diagram
-
-
-
-
?
benzoate + NADH + O2
?
show the reaction diagram
-
-
-
-
?
benzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
cinnamic acid + AH2 + O2
? + A + H2O
show the reaction diagram
Q6N8N2
good substrate of enzyme mutant F185L, and F185G, low activity with enzyme mutants F185V, F185I, F185A F185S, and F185T, no activity with the wild-type enzyme and mutants F185Y and F185W with cinnamic acid
-
-
?
N,N'-dimethyl-4-aminobenzoate + NADH + O2
4-aminobenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
N-methyl-4-aminobenzoate + NADH + O2
4-aminobenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
-
N-methyl-4-aminobenzoate + NADH + O2
4-aminobenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
veratrate + AH2 + O2
?
show the reaction diagram
Q2IU02
-
-
-
?
L-perillic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
substrate binding induces a large type I spin-state shift, highest shifts are observed with benzoic acids and benzaldehydes containing a substitutent at the 4-position. Palustrisredoxin A is the natural electron transfer cofactor protein
-
-
-
additional information
?
-
-
CYP199A2 shows a strong preference for para-substituted benzoate over identically substituted ortho- and meta- benzoates, and para-substituted benzenes, benzyl alcohols and benzaldehydes, a cytochrome P450 enzyme, the substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues, substrate binding and substrate channeling mechanism and structure, overview
-
-
-
additional information
?
-
-
CYP199A2 is a heme monooxygenase that catalyses the oxidation of para-substituted benzoic acids, the hydroxylation and desaturation of 4-ethylbenzoic acid, and thus may play a role in lignin degradation, CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin, Pux, and a flavoprotein palustrisredoxin reductase, PuR. Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system, overview
-
-
-
additional information
?
-
-
the enzyme performs regioselective oxidation of indole- and quinolinecarboxylic acids, it oxidizes 2-naphthoic acid and 4-ethylbenzoic acid, substrate specificity and regioselectivity of CYP199A2, overview. CYP199A2 does not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidizes indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid is converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generates only one product, 2-indolinone-6-carboxylic acid. The oxidation product of quinoline-6-carboxylic acid is 3-hydroxyquinoline-6-carboxylic acid
-
-
-
additional information
?
-
Q2IU02
CYP199A2 oxidizes para-substituted benzoic acids with almost total NADH-to-product conversion with the highest activity being observed in the oxidative demethylation of 4-methoxybenzoate. Exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product, exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product
-
-
-
additional information
?
-
Q6N8N2
substrate specficities of wild-type and F185 mutants, overview. The enzyme exhibits oxidation activity for aromatic carboxylic acids, including 2-naphthoic acid, 4-ethylbenzoic acid, and indole-and quinolinecarboxylic acids. No activity of the wild-type enzyme with cinnamic acid
-
-
-
additional information
?
-
Q2IU02
exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product
-
-
-
additional information
?
-
-
CYP199A2 is a heme monooxygenase that catalyses the oxidation of para-substituted benzoic acids, the hydroxylation and desaturation of 4-ethylbenzoic acid, and thus may play a role in lignin degradation, CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin, Pux, and a flavoprotein palustrisredoxin reductase, PuR. Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system, overview
-
-
-
additional information
?
-
Q6N8N2
CYP199A2 oxidizes para-substituted benzoic acids with almost total NADH-to-product conversion with the highest activity being observed in the oxidative demethylation of 4-methoxybenzoate. Exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product
-
-
-
additional information
additional information
-
-
-
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
additional information
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
uncoupling substrates are not oxygenized, NAD+ and H2O2 being the only products of the reaction
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
Q2IU02
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + AH2 + O2
4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
Q6N8N2
-
-
-
?
4-methoxybenzoate + NADH + O2
4-hydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + reduced ferredoxin + O2
4-hydroxybenzoate + formaldehyde + ferredoxin + H2O
show the reaction diagram
-
-
-
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
show the reaction diagram
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additional information
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CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin, Pux, and a flavoprotein palustrisredoxin reductase, PuR. Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system, overview
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-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FMN
-
prosthetic group of the oxidoreductase
heme
-
Phe at position 185 is situated directly above, and only 6.35 A from, the heme iron
NADH
-
requirement, can be replaced by NADPH with 40% efficiency
NADPH
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can replace NADH with 40% efficiency
palustrisredoxin
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CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin, Pux, and a flavoprotein palustrisredoxin reductase, PuR. Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system, overview. Interaction of CYP199A2 with PuxB mutants, structure of PuxB A105R, and with ferredoxin, detailed overview. RPA3956, PuxB, from strain CGA009 is a vertebrate-type [2Fe-2S] ferredoxin with the characteristic cysteine in ferredoxins involved in Fe-S cluster biogenesis, PuxB also supports substrate oxidation by CYP199A2
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palustrisredoxin
Q2IU02
i.e. Pux, RPA1872, with [2Fe-2S] cluster
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palustrisredoxin A
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-
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reduced ferredoxin
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-
additional information
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PuxB, 2Fe-2S ferredoxin, palustrisredoxin B, supports substrate oxidation by CYP199A2
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additional information
Q2IU02
the CYP enzyme surface closest to the heme (proximal surface) is the probable ferredoxin binding site; the CYP enzyme surface closest to the heme (proximal surface) is the probable ferredoxin binding site
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
-
both enzyme components are iron-sulfur proteins; mononuclear non-heme iron protein, 2Fe-2S cluster
Fe2+
-
both enzyme components are iron-sulfur proteins
Fe2+
-
[Fe2-S2]-cluster in the cofactors, overview
Fe2+
Q2IU02
heme enzyme; heme enzyme
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
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2,2'-dipyridyl
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3-Methoxybenzoate
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4-(2-Pyridylazo)resorcinol
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4-tert-Butylbenzoate
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competitive inhibitor of the O-demethylation of 3-nitro-4-methoxybenzoate, hinders O2-binding or O2-activation
4-trifluoromethylbenzoate
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5,5'-dithiobis(2-nitrobenzoate)
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8-hydroxyquinoline
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Bathocuproinedisulfonate
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bathophenanthrolinedisulfate
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preferentially inhibiting putidamonooxin
bathophenanthrolinedisulfate
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Benzoate
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competitive inhibition
Cl-
Q2IU02
addition of chloride to the phosphate buffered samples weakens substrate binding, chloride binding site structure, overview; addition of chloride to the phosphate buffered samples weakens substrate binding, chloride binding site structure, overview
Cumylhydroperoxide
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diethyldithiocarbamate
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Iodosobenzene
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Oxidized putidamonooxin
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60% inhibition with 3-nitro-4-methoxybenzoate as substrate, fully reactivated by Fe2+ and sulfhydryl-reagents
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p-chloromercuribenzoate
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p-chloromercuribenzoate
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reversible by GSH
Thenoyl trifluoroacetone
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Putidamonooxin
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essential part of the enzyme system, O2-activating 2Fe-2S-protein, identified by EPR and Mssbauer spectroscopy
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0003
3-Nitro-4-methoxybenzoate
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-
0.0007
3-Nitro-4-methoxybenzoate
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-
0.029
4-hydroxybenzoate
-
-
0.00007
4-Methoxybenzoate
-
-
0.0014
4-Methoxybenzoate
-
-
0.0058
4-methylaminobenzoate
-
-
0.0062
4-Methylbenzoate
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reaction in D2O
0.009
4-Methylbenzoate
-
-
0.009
4-Methylbenzoate
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reaction in H2O
0.0144
4-trifluoromethylbenzoate
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reaction in D2O
0.024
4-trifluoromethylbenzoate
-
-
0.024
4-trifluoromethylbenzoate
-
reaction in H2O
0.0367
Benzoate
-
reaction in H2O
0.077
N-methyl-4-aminobenzoate
-
-
0.00063
NADH
-
reconstituted enzyme
0.008
NADH
-
reductase
0.14
NADPH
-
reconstituted enzyme
0.0019
O2
-
+ 4-methoxybenzoate
0.0038
O2
-
+ 3,4-dimethoxybenzoate
0.01
O2
-
+ 4-ethoxybenzoate
0.051
O2
-
+ N-methyl-4-aminobenzoate
0.055
O2
-
+ benzoate
0.055
O2
-
+ 4-methylbenzoate
0.03
Putidamonooxin
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-
-
0.055
Benzoate
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reaction in D2O
additional information
additional information
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steady-state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
37.9
4-Methoxybenzoate
Q2IU02
pH 7.4, 30C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.002
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4-hyroxybenzoate, reductase
0.004
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4-hydroxybenzoate, monooxygenase
0.008
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3-methoxybenzoate, 4-methoxybenzoate, cell-free extract
0.009
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N,N-dimethyl-4-aminobenzoate
0.018
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superoxide anion instead of substrate, putidamonooxin
0.021
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4-hydroxy-3-methoxybenzoate
0.023
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3-hydroxybenzoate, reconstituted enzyme
0.024
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4-ethoxybenzoate, N-methyl-4-aminobenzoate
0.025
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3,4-dimethoxybenzoate
0.04
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4-methoxybenzoate, cell-free extract
0.055
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4-hyxdroxybenzoate
0.077
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4-hydroxybenzoate, reconstituted enzyme
0.11
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3-methoxybenzoate
0.178
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N-methyl-4-aminobenzoate
0.192
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3-nitro-4-methoxybenzoate, putidamonooxin
0.27
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4-methoxybenzoate, putidamonooxin
0.314
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4-ethoxybenzoate, piperonylate
0.345
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3,4-dimethoxybenzoate
0.36
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4-methoxybenzoate
21.96
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4-methoxybenzoate
194.5
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3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in H2O
207.8
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4-trifluoromethylbenzoate, oxygen uptake, reaction in D2O
239.8
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3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in D2O
265.6
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benzoate, oxygen uptake, reaction in D2O
295.1
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benzoate, oxygen uptake, reaction in H2O
303.9
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3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in H2O
305.2
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4-methylbenzoate, NADH oxidation, reaction in H2O
310.1
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3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in D2O; 3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in H2O
315.5
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4-trifluoromethylbenzoate, NADH oxidation, reaction in D2O
317.2
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4-methylbenzoate, NADH oxidation, reaction in D2O
317.4
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3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in D2O
326.7
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3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in H2O
329.2
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3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in D2O
339.1
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benzoate, NADH oxidation, reaction in D2O
340.2
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3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in H2O
345
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4-trifluoromethylbenzoate, oxygen uptake, reaction in H2O
345.6
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benzoate, NADH oxidation, reaction in H2O
353.5
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3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in D2O
360.4
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3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in H2O
367
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4-methylbenzoate, oxygen uptake, reaction in H2O
368
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4-methylbenzoate, oxygen uptake, reaction in D2O
380.2
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3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in D2O
391.5
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4-methoxybenzoate, oxygen uptake, reaction in D2O
394.1
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4-methoxybenzoate, oxygen uptake, reaction in H2O
402.6
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3-nitro-4-methoxybenzoate, NADH oxidation, reaction in D2O
409.7
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4-trifluoromethylbenzoate, NADH oxidation, reaction in H2O
422.1
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3-nitro-4-methoxybenzoate, NADH oxidation, reaction in H2O
432.4
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3-nitro-4-methoxybenzoate, oxygen uptake, reaction in H2O
432.7
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3-nitro-4-methoxybenzoate, oxygen uptake, reaction in D2O
442.2
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4-methoxybenzoate, NADH oxidation, reaction in D2O
445
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4-methoxybenzoate, NADH oxidation, reaction in H2O
450.9
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3-nitro-4-methoxybenzoate, product formation, reaction in H2O
487.3
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3-nitro-4-methoxybenzoate, product formation, reaction in D2O
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
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assay at; the electron transfer to CYP199A2 is studied
7.4
Q2IU02
assay at; assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.3 - 9
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about half-maximal activity at pH 7.3 and 9.0, reductase
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
Q2IU02
assay at; assay at
PDB
SCOP
CATH
ORGANISM
Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009)
Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
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alpha2, 2 * 52000, putidamonooxin, SDS-PAGE
oligomer
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3-4 * 41500, putidamonooxin, SDS-PAGE
oligomer
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3-4 * 33000-45000, putidamonooxin, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
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30% carbohydrate in each of the 50000 Da subunits
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme CYP199A2 bound to substrate 4-methoxybenzoate, X-ray diffraction structure determination and analysis at 1.8 A resolution; enzyme CYP199A4 free and bound to substrate 4-methoxybenzoate, hanging drop vapour diffusion method, for the free enzyme: mixing of 0.001 ml of protein solution containing 50 mg/ml protein in 20 mM HEPES, pH 7.4, 150 mM KCl, 1 mM DTT, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 1.45-1.5 M ammonium sulfate, and 0.1 M sodium chloride, and equilibration against 0.2 ml of reservoir solution, 20C, 1 week, for the substrate-bound enzyme: mixing of 0.001 ml of protein solution containing 40 mg/ml protein in 20 mM HEPES, pH 7.4, 150 mM KCl, and 10 mM 2-mercaptoethanol and saturated with 4-methoxybenzoate, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 1.45 M ammonium sulfate, and 0.1 M sodium chloride, and equilibration against 0.2 ml of reservoir solution, 20C, 2 weeks, X-ray diffraction structure determination and analysis at 2.6 A and 2.0 A resolution, respectively
Q2IU02
purified recombinant CYP199A2, 16 C, the hanging drop vapor diffusion method under aerobic conditions, 0.0015 ml of protein solution is mixed with 0.0015 ml of reservoir solution, addition of 200 ml reservoir solution, containing 15% PEG 4000, 100 mM sodium citrate pH 5.6, 20% isopropanol with 4% v/v t-butanol, 1 week, X-ray diffraction structure deternnation and analysis at 2.0 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
4-methoxybenzoate, stabilizes
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ethanol, 5-15% v/v, stabilizes activity in buffer and crude extract
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NADH, not NADPH or substrate stabilize the reductase
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substrate or substrate analogues stabilize putidamomooxin by preventing loss of Fe2+
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
aerobic conditions, 0-4C, 50% activity lost within 24 h
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438344
putidamonooxin and NADH-reductase, extremely O2-sensitive, GSH, DTT, 2-mercaptoethanol prevent putidamonooxin oxidation of putdamonooxin
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438346
putidamonooxin and NADH-reductase, extremely O2-sensitive, purification and storage of the reductase under anaerobic conditions
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438350
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 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
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0-4C, more than 24 days stable in crude extract under anaerobic conditions
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4C, concentrated putidamonooxin stable for several months in N2-atmosphere with DTT and dithionite
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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recombinant enzyme from Escherichia coli strain BL21(DE3) by two different steps of anion exchange chromatography, followed by gel filtration; recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by two different steps of anion exchange chromatography, followed by gel filtration
Q2IU02
recombinant His-tagged enzyme from Escherichia coli BL21(DE3) by nickel affinity chromatography and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CYP199A2 gene RPA1871, coexpression with palustris redoxin gene from Rhodopseudomonas palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli
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expression in Escherichia coli strain BL21(DE3); wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
Q2IU02
expression of wild-type and mutant CYP199A2 in Escherichia coli strain BL21(DE3)
-
overexpression of the His-tagged enzyme in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F185A
-
site-directed mutagenesis, the mutant shows activity with cinnamic acid in contrast to the wild-type enzyme
F185G
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site-directed mutagenesis, the mutant shows activity with cinnamic acid in contrast to the wild-type enzyme
F185I
-
site-directed mutagenesis, the mutant shows activity with cinnamic acid in contrast to the wild-type enzyme
F185I
Q2IU02
site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
F185L
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site-directed mutagenesis, the mutant shows activity with cinnamic acid in contrast to the wild-type enzyme, the F185L mutant exhibits 5.5times higher hydroxylation activity for 4-coumaric acid than the wild-type enzyme
F185S
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site-directed mutagenesis, the mutant shows reduced activity with 2-naphthoic acid compared to the wild-type enzyme
F185T
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site-directed mutagenesis, the mutant shows reduced activity with 2-naphthoic acid compared to the wild-type enzyme
F185V
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site-directed mutagenesis, the mutant shows activity with cinnamic acid in contrast to the wild-type enzyme
F185V
Q2IU02
site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 35% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
F185W
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site-directed mutagenesis, inactive mutant
R243T
Q2IU02
site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
R92E
Q2IU02
site-directed mutagenesis, the spin state shift is similar to the wild-type enzyme, but the mutant shows 3fold higher KD for the substrate, NADH consumption is reduced 9fold compared to the wild-type enzyme
S95V
Q2IU02
site-directed mutagenesis, the mutation abolishes the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate and results in a 99% drop in the NADH consumption rate comared to the wild-type enzyme
F185I
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site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
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F185V
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site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 35% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
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R243T
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site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation
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R92E
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site-directed mutagenesis, the spin state shift is similar to the wild-type enzyme, but the mutant shows 3fold higher KD for the substrate, NADH consumption is reduced 9fold compared to the wild-type enzyme
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S95V
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site-directed mutagenesis, the mutation abolishes the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate and results in a 99% drop in the NADH consumption rate comared to the wild-type enzyme
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F185Y
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site-directed mutagenesis, inactive mutant
additional information
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substrate specficities of wild-type and F185 mutants, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
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CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids