Information on EC 1.14.13.128 - 7-methylxanthine demethylase

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The expected taxonomic range for this enzyme is: Pseudomonas putida

EC NUMBER
COMMENTARY hide
1.14.13.128
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RECOMMENDED NAME
GeneOntology No.
7-methylxanthine demethylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
7-methylxanthine + O2 + NAD(P)H + H+ = xanthine + NAD(P)+ + H2O + formaldehyde
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
caffeine degradation III (bacteria, via demethylation)
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Caffeine metabolism
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
7-methylxanthine:oxygen oxidoreductase (demethylating)
A non-heme iron oxygenase. The enzyme from the bacterium Pseudomonas putida prefers NADH over NADPH. The enzyme is specific for 7-methylxanthine [2]. Forms part of the caffeine degradation pathway.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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catabolism of caffeine in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline, whereas theobromine is the major metabolite in bacteria
metabolism
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catabolism of caffeine in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline, whereas theobromine is the major metabolite in bacteria. Catabolism of caffeine in microorganisms, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,3,7-trimethylxanthine + O2 + NAD(P)H + H+
3,7-dimethylxanthine + formaldehyde + NAD(P)+
show the reaction diagram
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i.e. caffeine
i.e. theobromine
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1,3,7-trimethylxanthine + O2 + NADPH + H+
1,3-dimethylxanthine + formaldehyde + NADP+ + H2O
show the reaction diagram
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i.e. caffeine
i.e. theophylline
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1,3,7-trimethylxanthine + O2 + NADPH + H+
1,7-dimethylxanthine + formaldehyde + NADP+ + H2O
show the reaction diagram
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i.e. caffeine
i.e. paraxanthine
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1,7-dimethylxanthine + 2 O2 + 2 NADH + 2 H+
xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde
show the reaction diagram
1,7-dimethylxanthine + 2 O2 + 2 NADPH + 2 H+
xanthine + 2 NADP+ + 2 H2O + 2 formaldehyde
show the reaction diagram
1,7-dimethylxanthine + O2 + NADH + H+
7-methylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
1,7-dimethylxanthine + O2 + NADPH + H+
7-methylxanthine + NADP+ + H2O + formaldehyde
show the reaction diagram
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i.e. paraxanthine, activity of the reductase component of the N-demethylase holoenzyme (Ccr) with NADPH is 22% of that with NADH. The enzyme also catalyzes the further demethylation of the product 7-methylxanthine to xanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
3,7-dimethylxanthine + O2 + NAD(P)H + H+
monomethylxanthine + formaldehyde + NADP+
show the reaction diagram
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theobromine demethylase
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-
?
3-methylxanthine + O2 + NADH + H+
xanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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3-methylxanthine demethylation is 12% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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-
?
7-methylxanthine + O2 + NAD(P)H + H+
xanthine + formaldehyde + NADP+
show the reaction diagram
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heteroxanthine demethylase, substrate-selective
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-
?
7-methylxanthine + O2 + NAD(P)H + H+
xanthine + NAD(P)+ + H2O + formaldehyde
show the reaction diagram
7-methylxanthine + O2 + NADH + H+
xanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
7-methylxanthine + O2 + NADPH + H+
xanthine + NADP+ + H2O + formaldehyde
show the reaction diagram
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-
-
-
?
caffeine + 2 O2 + 2 NADH + 2 H+
7-methylxanthine + 2 NAD+ + 2 H2O + 2 formaldehyde
show the reaction diagram
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i.e. 1,3,7-trimethylxanthine. Caffeine demethylation is 7% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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-
?
caffeine + 3 O2 + 3 NADH + 3 H+
xanthine + 3 NAD+ + 3 H2O + 3 formaldehyde
show the reaction diagram
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i.e. 1,3,7-trimethylxanthine. Caffeine demethylation is 7% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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-
?
caffeine + O2 + NADH + H+
1,7-dimethylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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i.e. 1,3,7-trimethylxanthine. Caffeine demethylation is 7% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
caffeine + O2 + NADH + H+
theobromine + NAD+ + H2O + formaldehyde
show the reaction diagram
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i.e. 1,3,7-trimethylxanthine. Caffeine demethylation is 7% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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-
?
theobromine + 2 O2 + 2 NADH + 2 H+
xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde
show the reaction diagram
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i.e. 3,7-dimethylxanthine. Theobromine demethylation is 13% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
theobromine + O2 + NADH + H+
3-methylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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i.e. 3,7-dimethylxanthine. Theobromine demethylation is 13% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
theobromine + O2 + NADH + H+
7-methylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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i.e. 3,7-dimethylxanthine. Theobromine demethylation is 13% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
theophylline + 2 O2 + 2 NADH + 2 H+
xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde
show the reaction diagram
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1,3-dimethylxanthine. Theophylline demethylation is 3% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
theophylline + O2 + NADH + H+
1-methylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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1,3-dimethylxanthine. Theophylline demethylation is 3% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
theophylline + O2 + NADH + H+
3-methylxanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
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1,3-dimethylxanthine. Theophylline demethylation is 3% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
1,3,7-trimethylxanthine + O2 + NAD(P)H + H+
3,7-dimethylxanthine + formaldehyde + NAD(P)+
show the reaction diagram
-
i.e. caffeine
i.e. theobromine
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1,3,7-trimethylxanthine + O2 + NADPH + H+
1,3-dimethylxanthine + formaldehyde + NADP+ + H2O
show the reaction diagram
-
i.e. caffeine
i.e. theophylline
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1,3,7-trimethylxanthine + O2 + NADPH + H+
1,7-dimethylxanthine + formaldehyde + NADP+ + H2O
show the reaction diagram
-
i.e. caffeine
i.e. paraxanthine
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7-methylxanthine + O2 + NAD(P)H + H+
xanthine + NAD(P)+ + H2O + formaldehyde
show the reaction diagram
7-methylxanthine + O2 + NADH + H+
xanthine + NAD+ + H2O + formaldehyde
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)H
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dependent on
NADPH
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activity of Ccr (reductase component of the N-demethylase holoenzyme) with NADPH is 22% of that with NADH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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1 mM, activation to 123% of control
Fe2+
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preincubation of Ndm for 15 min with 1 mM Fe2+, followed by desalting, increases the iron content to 20.1 mol per mol hexameric N-demethylase component (Ndm) of the N-demethylase holoenzyme. After this treatment, Ndm specific activity increases about 6fold (when 50 mM Fe2+ is present in the enzyme reaction mixture). N-Demethylase component (Ndm) of the N-demethylase holoenzyme is deduced to be a Rieske [2Fe-2S]-domain containing non-haem iron oxygenase
Mg2+
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1 mM, activation to 132% of control
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
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0.2 mM, complete inhibition
Co2+
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0.2 mM, 40% inhibition
Cu2+
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0.2 mM, complete inhibition
Fe2+
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1 mM, 37% inhibition
Hg2+
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1 mM, 98% inhibition
Sn2+
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0.2 mM, 17% inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0504
1,7-dimethylxanthine
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pH 7.5, 30C, kinetic parameters for two-subunit N-demethylase component (Ndm), in the presence of saturating amounts of Ccr (reductase component with cytochrome c reductase activity) and 50 mM Fe2+
1.1
3,7-Dimethylxanthine
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theobromine demethylase, pH and temperature not specified in the publication
0.0153 - 0.0638
7-methylxanthine
0.113
O2
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pH and temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.27
1,7-dimethylxanthine
Pseudomonas putida
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pH 7.5, 30C, kinetic parameters for two-subunit N-demethylase component (Ndm), in the presence of saturating amounts of Ccr (reductase component with cytochrome c reductase activity) and 50 mM Fe2+
0.16 - 1.58
7-methylxanthine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.4
1,7-dimethylxanthine
Pseudomonas putida
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pH 7.5, 30C, kinetic parameters for two-subunit N-demethylase component (Ndm), in the presence of saturating amounts of Ccr (reductase component with cytochrome c reductase activity) and 50 mM Fe2+
6872
10.17 - 24.8
7-methylxanthine
2307
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55.4
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pH 7.5, 30C, activity with paraxanthine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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caffeine demethylase
7.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
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about 50% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22 - 24
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caffeine demethylase
30
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assay at
30 - 35
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
240000
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gel filtration, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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1 min, complete inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stabilization can be achieved by addition of either glycerol (20%), bovine serum albumin (10 mg/ml), dithiothreitol (1 mM) or the substrate 7-methylxanthine (1 mM). Storage under nitrogen atmosphere has a stabilizing influence. The addition of caffeine (1 mM), dimethylxanthines (1 mM) and protease inhibitors (pepstatin A, PMSF, 0.1 mM) is without influence on stability
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme is stable for over 1 month
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4C, 5 days, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme is stabel for at lewast 5 days
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4C, pH 6.0, 94% loss of activity after 24 h
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similar to caffeine demethylase, heteroxanthinedemethylase is also unstable and loss of activity occurs upon storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native heteroxanthine demethylase partially
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Ni-NTA column chromatography
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Ni-NTA column chromatography and Sephacryl S200 gel filtration
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purification of two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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methylxanthine intermediates of caffeine catabolism obtained by the action of N-demethylases have many applications. In medicine, theobromine and theophylline are used as diuretics, vasodilators, and myocardial stimulants. Monomethylxanthines can be converted to effective caffeine derivatives by chemical derivatization and hence can serve as interesting alternatives to caffeine. Xanthine also finds pharmaceutical application in drugs for treatment of asthma. The biotechnological potential of N-demethylases therefore lies not only in general decaffeination purposes but also in specific product recovery from caffeine
pharmacology
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methylxanthine intermediates of caffeine catabolism obtained by the action of N-demethylases have many applications. In medicine, theobromine and theophylline are used as diuretics, vasodilators, and myocardial stimulants. Monomethylxanthines can be converted to effective caffeine derivatives by chemical derivatization and hence can serve as interesting alternatives to caffeine. Xanthine also finds pharmaceutical application in drugs for treatment of asthma. The biotechnological potential of N-demethylases therefore lies not only in general decaffeination purposes but also in specific product recovery from caffeine