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Literature summary for 1.14.13.128 extracted from

  • Summers, R.M.; Louie, T.M.; Yu, C.L.; Subramanian, M.
    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.
    View publication on PubMed

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 [┬Ámol/min/mg] Specific Activity Maximum [┬Ámol/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