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Literature summary for 1.14.13.128 extracted from
- Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source (2011), Microbiology, 157, 583-592.
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0504 | - |
1,7-dimethylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida |  |
| 0.0638 | - |
7-methylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | 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 | Pseudomonas putida | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 240000 | - |
gel filtration, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme | Pseudomonas putida |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 7-methylxanthine + O2 + NAD(P)H + H+ | Pseudomonas putida | part of the caffeine degradation pathway in Pseudomonas putida | xanthine + NAD(P)+ + H2O + formaldehyde | - |
? | |
| 7-methylxanthine + O2 + NAD(P)H + H+ | Pseudomonas putida CBB5 | part of the caffeine degradation pathway in Pseudomonas putida | xanthine + NAD(P)+ + H2O + formaldehyde | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas putida | - |
- |
- |
| Pseudomonas putida CBB5 | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| purification of two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme | Pseudomonas putida |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 55.4 | - |
pH 7.5, 30°C, activity with paraxanthine | Pseudomonas putida |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme is stable for over 1 month | Pseudomonas putida |
| 4°C, 5 days, two-subunit N-demethylase component (Ndm) of the N-demethylase holoenzyme is stabel for at lewast 5 days | Pseudomonas putida |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 1,7-dimethylxanthine + 2 O2 + 2 NADH + 2 H+ | i.e. paraxanthine, NADH is the preferred cofactor of reductase component of the N-demethylase holoenzyme (Ccr). The product 7-methylxanthine is further demethylated to xanthine. 1,7-Dimethylxanthine (paraxanthine) demethylation is 22% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida | xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde | - |
? | |
| 1,7-dimethylxanthine + 2 O2 + 2 NADH + 2 H+ | i.e. paraxanthine, NADH is the preferred cofactor of reductase component of the N-demethylase holoenzyme (Ccr). The product 7-methylxanthine is further demethylated to xanthine. 1,7-Dimethylxanthine (paraxanthine) demethylation is 22% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida CBB5 | xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde | - |
? | |
| 1,7-dimethylxanthine + 2 O2 + 2 NADPH + 2 H+ | 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 | Pseudomonas putida | xanthine + 2 NADP+ + 2 H2O + 2 formaldehyde | - |
? | |
| 1,7-dimethylxanthine + 2 O2 + 2 NADPH + 2 H+ | 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 | Pseudomonas putida CBB5 | xanthine + 2 NADP+ + 2 H2O + 2 formaldehyde | - |
? | |
| 1,7-dimethylxanthine + O2 + NADH + H+ | i.e. paraxanthine, NADH is the preferred cofactor of reductase component of the N-demethylase holoenzyme (Ccr). The product 7-methylxanthine is further demethylated to xanthine. 1,7-Dimethylxanthine (paraxanthine) demethylation is 22% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida | 7-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| 1,7-dimethylxanthine + O2 + NADH + H+ | i.e. paraxanthine, NADH is the preferred cofactor of reductase component of the N-demethylase holoenzyme (Ccr). The product 7-methylxanthine is further demethylated to xanthine. 1,7-Dimethylxanthine (paraxanthine) demethylation is 22% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida CBB5 | 7-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| 1,7-dimethylxanthine + O2 + NADPH + H+ | 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 | Pseudomonas putida | 7-methylxanthine + NADP+ + H2O + formaldehyde | - |
? | |
| 3-methylxanthine + O2 + NADH + H+ | 3-methylxanthine demethylation is 12% of the activity compared to demethylation of 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida | xanthine + NAD+ + H2O + formaldehyde | - |
? | |
| 7-methylxanthine + O2 + NAD(P)H + H+ | part of the caffeine degradation pathway in Pseudomonas putida | Pseudomonas putida | xanthine + NAD(P)+ + H2O + formaldehyde | - |
? | |
| 7-methylxanthine + O2 + NAD(P)H + H+ | part of the caffeine degradation pathway in Pseudomonas putida | Pseudomonas putida CBB5 | xanthine + NAD(P)+ + H2O + formaldehyde | - |
? | |
| 7-methylxanthine + O2 + NADH + H+ | the enzyme most actively demethylates 7-methylxanthine. Activity is absolutely dependent of oxygen as a cosubstrate | Pseudomonas putida | xanthine + NAD+ + H2O + formaldehyde | - |
? | |
| caffeine + 2 O2 + 2 NADH + 2 H+ | 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 | Pseudomonas putida | 7-methylxanthine + 2 NAD+ + 2 H2O + 2 formaldehyde | - |
? | |
| caffeine + 3 O2 + 3 NADH + 3 H+ | 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 | Pseudomonas putida | xanthine + 3 NAD+ + 3 H2O + 3 formaldehyde | - |
? | |
| caffeine + O2 + NADH + H+ | 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 | Pseudomonas putida | 1,7-dimethylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| caffeine + O2 + NADH + H+ | 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 | Pseudomonas putida | theobromine + NAD+ + H2O + formaldehyde | - |
? | |
| additional information | Ndm exhibits broad-based activity towards caffeine, theophylline, theobromine, 7-methylxanthine and 3-methylxanthine, all of which are growth substrates for Pseudomonas putida CBB5. Production of xanthine from all of these methylxanthines is confirmed. Ndm is most active on 7-methylxanthine, followed by 1,7-dimethylxanthine (paraxanthine), theobromine, 3-methylxanthine, caffeine and theophylline. Ndm does not catalyse O-demethylation of vanillate or vanillin, even after prolonged incubation | Pseudomonas putida | ? | - |
? | |
| additional information | Ndm exhibits broad-based activity towards caffeine, theophylline, theobromine, 7-methylxanthine and 3-methylxanthine, all of which are growth substrates for Pseudomonas putida CBB5. Production of xanthine from all of these methylxanthines is confirmed. Ndm is most active on 7-methylxanthine, followed by 1,7-dimethylxanthine (paraxanthine), theobromine, 3-methylxanthine, caffeine and theophylline. Ndm does not catalyse O-demethylation of vanillate or vanillin, even after prolonged incubation | Pseudomonas putida CBB5 | ? | - |
? | |
| theobromine + 2 O2 + 2 NADH + 2 H+ | 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 | Pseudomonas putida | xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde | - |
? | |
| theobromine + O2 + NADH + H+ | 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 | Pseudomonas putida | 3-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| theobromine + O2 + NADH + H+ | 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 | Pseudomonas putida | 7-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| theophylline + 2 O2 + 2 NADH + 2 H+ | 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 | Pseudomonas putida | xanthine + 2 NAD+ + 2 H2O + 2 formaldehyde | - |
? | |
| theophylline + O2 + NADH + H+ | 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 | Pseudomonas putida | 1-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
| theophylline + O2 + NADH + H+ | 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 | Pseudomonas putida | 3-methylxanthine + NAD+ + H2O + formaldehyde | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | the soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm), the native Ndm enzyme is probably composed of the two subunits (40000 Da (NdmA) and 35000 Da (NdmB)) in a hexameric configuration | Pseudomonas putida |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Pseudomonas putida |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.27 | - |
1,7-dimethylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida | |
| 1.58 | - |
7-methylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Pseudomonas putida |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6.5 | 8 | about 50% of maximal activity | Pseudomonas putida |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NADH | preferred cofactor of Ccr (reductase component of the N-demethylase holoenzyme) | Pseudomonas putida | |
| NADPH | activity of Ccr (reductase component of the N-demethylase holoenzyme) with NADPH is 22% of that with NADH | Pseudomonas putida | |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 5.4 | - |
1,7-dimethylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida | |
| 24.8 | - |
7-methylxanthine | pH 7.5, 30°C, 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+ | Pseudomonas putida | |
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