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 ACCESSION NO.
LITERATURE
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
-
4-methoxybenzoate + AH2 + O2 = 4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
a terminal oxygenase
-
4-methoxybenzoate + AH2 + O2 = 4-hydroxybenzoate + formaldehyde + A + H2O
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dealkylation
-
-
-
-
N-demethylation
-
-
-
-
O-demethylation
-
-
-
-
oxidation
-
-
-
-
oxidative demethylation
-
-
oxidative demethylation
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
S-demethylation
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Benzoate degradation
-
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 ACCESSION NO.
COMMENTARY
LITERATURE
4-methoxybenzoate 4-monooxygenase (O-demethylating)
-
-
-
-
4-methoxybenzoate monooxygenase
-
-
4-methoxybenzoate monooxygenase
-
-
-
4-methoxybenzoate O-demethylase
-
-
-
-
cytochrome P450 199A2
-
-
oxygenase, 4-methoxybenzoate 4-mono- (O-demethylating)
-
-
-
-
p-anisic O-demethylase
-
-
-
-
piperonylate-4-O-demethylase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37256-78-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
CGA009; gene RPA1871
-
-
Manually annotated by BRENDA team
isoform CYP199A2
-
-
Manually annotated by BRENDA team
CGA009; gene RPA1871
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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
-
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
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3,4-dimethoxybenzoate + NADH + O2
4-hydroxy-3-methoxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
-
-
-
?
3,4-methylenedioxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
piperonylate
protocatechuate
?
3,4-methylenedioxybenzoate + NADH + O2
3,4-dihydroxybenzoate + NAD+ + H2O + formaldehyde
show the reaction diagram
-
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
-
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
-
partial uncoupler
-
?
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
-
-
-
-
?
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
-
-
-
-
?
4-chlorobenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-ethoxybenzoate + NADH + O2
4-hydroxybenzoate + acetaldehyde + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
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
-
-
?
4-ethylbenzoic acid + NADH + O2
? + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-Hydroxy-3-methoxybenzoate + NADH + O2
?
show the reaction diagram
-
vanillate, partial uncoupler
-
-
?
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
-
-
-
?
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 + 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 putidaredoxin + O2
4-hydroxybenzoate + formaldehyde + oxidized putidaredoxin + H2O
show the reaction diagram
-
very low activity with putidaredoxin
-
-
?
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
-
-
-
-
?
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
-
-
-
-
?
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
?
-
-
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
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 ACCESSION NO.
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 + 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
-
-
-
-
?
additional information
?
-
-
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
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
FMN
-
prosthetic group of the oxidoreductase
NADH
-
requirement, can be replaced by NADPH with 40% efficiency
NADPH
-
can replace NADH with 40% efficiency
palustrisredoxin
-
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
-
palustrisredoxin A
-
-
-
reduced ferredoxin
-
-
additional information
-
PuxB, 2Fe-2S ferredoxin, palustrisredoxin B, supports substrate oxidation by CYP199A2
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
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
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
-
-
2,2'-dipyridyl
-
-
3-Methoxybenzoate
-
-
4-(2-Pyridylazo)resorcinol
-
-
4-tert-Butylbenzoate
-
competitive inhibitor of the O-demethylation of 3-nitro-4-methoxybenzoate, hinders O2-binding or O2-activation
4-trifluoromethylbenzoate
-
-
5,5'-dithiobis(2-nitrobenzoate)
-
-
8-hydroxyquinoline
-
-
Bathocuproinedisulfonate
-
-
bathophenanthrolinedisulfate
-
preferentially inhibiting putidamonooxin
bathophenanthrolinedisulfate
-
-
Benzoate
-
competitive inhibition
Cumylhydroperoxide
-
-
diethyldithiocarbamate
-
-
Iodosobenzene
-
-
Oxidized putidamonooxin
-
60% inhibition with 3-nitro-4-methoxybenzoate as substrate, fully reactivated by Fe2+ and sulfhydryl-reagents
-
p-chloromercuribenzoate
-
-
p-chloromercuribenzoate
-
reversible by GSH
Thenoyl trifluoroacetone
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Putidamonooxin
-
essential part of the enzyme system, O2-activating 2Fe-2S-protein, identified by EPR and Mössbauer spectroscopy
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0003
-
3-Nitro-4-methoxybenzoate
-
-
0.0007
-
3-Nitro-4-methoxybenzoate
-
-
0.029
-
4-hydroxybenzoate
-
-
0.00007
-
4-Methoxybenzoate
-
-
0.0014
-
4-Methoxybenzoate
-
-
0.0058
-
4-methylaminobenzoate
-
-
0.0062
-
4-Methylbenzoate
-
reaction in D2O
0.009
-
4-Methylbenzoate
-
-
0.009
-
4-Methylbenzoate
-
reaction in H2O
0.0144
-
4-trifluoromethylbenzoate
-
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
-
-
-
0.055
-
Benzoate
-
reaction in D2O
additional information
-
additional information
-
steady-state kinetics
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.002
-
-
4-hyroxybenzoate, reductase
0.004
-
-
4-hydroxybenzoate, monooxygenase
0.008
-
-
3-methoxybenzoate, 4-methoxybenzoate, cell-free extract
0.009
-
-
N,N-dimethyl-4-aminobenzoate
0.018
-
-
superoxide anion instead of substrate, putidamonooxin
0.021
-
-
4-hydroxy-3-methoxybenzoate
0.023
-
-
3-hydroxybenzoate, reconstituted enzyme
0.024
-
-
4-ethoxybenzoate, N-methyl-4-aminobenzoate
0.025
-
-
3,4-dimethoxybenzoate
0.04
-
-
4-methoxybenzoate, cell-free extract
0.055
-
-
4-hyxdroxybenzoate
0.077
-
-
4-hydroxybenzoate, reconstituted enzyme
0.11
-
-
3-methoxybenzoate
0.178
-
-
N-methyl-4-aminobenzoate
0.192
-
-
3-nitro-4-methoxybenzoate, putidamonooxin
0.27
-
-
4-methoxybenzoate, putidamonooxin
0.314
-
-
4-ethoxybenzoate, piperonylate
0.345
-
-
3,4-dimethoxybenzoate
0.36
-
-
4-methoxybenzoate
21.96
-
-
4-methoxybenzoate
194.5
-
-
3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in H2O
207.8
-
-
4-trifluoromethylbenzoate, oxygen uptake, reaction in D2O
239.8
-
-
3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in D2O
265.6
-
-
benzoate, oxygen uptake, reaction in D2O
295.1
-
-
benzoate, oxygen uptake, reaction in H2O
303.9
-
-
3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in H2O
305.2
-
-
4-methylbenzoate, NADH oxidation, reaction in H2O
310.1
-
-
3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in D2O; 3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in H2O
315.5
-
-
4-trifluoromethylbenzoate, NADH oxidation, reaction in D2O
317.2
-
-
4-methylbenzoate, NADH oxidation, reaction in D2O
317.4
-
-
3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in D2O
326.7
-
-
3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in H2O
329.2
-
-
3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in D2O
339.1
-
-
benzoate, NADH oxidation, reaction in D2O
340.2
-
-
3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in H2O
345
-
-
4-trifluoromethylbenzoate, oxygen uptake, reaction in H2O
345.6
-
-
benzoate, NADH oxidation, reaction in H2O
353.5
-
-
3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in D2O
360.4
-
-
3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in H2O
367
-
-
4-methylbenzoate, oxygen uptake, reaction in H2O
368
-
-
4-methylbenzoate, oxygen uptake, reaction in D2O
380.2
-
-
3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in D2O
391.5
-
-
4-methoxybenzoate, oxygen uptake, reaction in D2O
394.1
-
-
4-methoxybenzoate, oxygen uptake, reaction in H2O
402.6
-
-
3-nitro-4-methoxybenzoate, NADH oxidation, reaction in D2O
409.7
-
-
4-trifluoromethylbenzoate, NADH oxidation, reaction in H2O
422.1
-
-
3-nitro-4-methoxybenzoate, NADH oxidation, reaction in H2O
432.4
-
-
3-nitro-4-methoxybenzoate, oxygen uptake, reaction in H2O
432.7
-
-
3-nitro-4-methoxybenzoate, oxygen uptake, reaction in D2O
442.2
-
-
4-methoxybenzoate, NADH oxidation, reaction in D2O
445
-
-
4-methoxybenzoate, NADH oxidation, reaction in H2O
450.9
-
-
3-nitro-4-methoxybenzoate, product formation, reaction in H2O
487.3
-
-
3-nitro-4-methoxybenzoate, product formation, reaction in D2O
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.4
-
-
assay at; the electron transfer to CYP199A2 is studied
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.3
9
-
about half-maximal activity at pH 7.3 and 9.0, reductase
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimer
-
alpha2, 2 * 52000, putidamonooxin, SDS-PAGE
oligomer
-
3-4 * 41500, putidamonooxin, SDS-PAGE
oligomer
-
3-4 * 33000-45000, putidamonooxin, SDS-PAGE; 3-4 * 41500, putidamonooxin, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycoprotein
-
30% carbohydrate in each of the 50000 Da subunits
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purified recombinant CYP199A2, 16 ºC, 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
-
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4-methoxybenzoate, stabilizes
-
ethanol, 5-15% v/v, stabilizes activity in buffer and crude extract
-
NADH, not NADPH or substrate stabilize the reductase
-
substrate or substrate analogues stabilize putidamomooxin by preventing loss of Fe2+
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
aerobic conditions, 0-4°C, 50% activity lost within 24 h
-
438344
putidamonooxin and NADH-reductase, extremely O2-sensitive, GSH, DTT, 2-mercaptoethanol prevent putidamonooxin oxidation of putdamonooxin
-
438346
putidamonooxin and NADH-reductase, extremely O2-sensitive, purification and storage of the reductase under anaerobic conditions
-
438350
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-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
-
0-4°C, more than 24 days stable in crude extract under anaerobic conditions
-
4°C, concentrated putidamonooxin stable for several months in N2-atmosphere with DTT and dithionite
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme
-
recombinant His-tagged enzyme from Escherichia coli BL21(DE3) by nickel affinity chromatography and gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
overexpression of the His-tagged enzyme in Escherichia coli BL21(DE3)
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids