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Literature summary extracted from

  • Kim, J.H.; Kim, B.H.; Brooks, S.; Kang, S.Y.; Summers, R.M.; Song, H.K.
    Structural and mechanistic insights into caffeine degradation by the bacterial N-demethylase complex (2019), J. Mol. Biol., 431, 3647-3661 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.B34 gene ndmD, recombinant overexpression of N-terminally MBP-tagged NdmD, coexpression with NdmC and NdmE Pseudomonas putida
1.14.13.128 gene ndmC, recombinant overexpression of His-tagged wild-type and mutant NdmC proteins, coexpression with NdmD and NdmE Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.128 H120A/D237A site-directed mutagenesis, catalytically inactive mutant Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.128 7-methylxanthine + O2 + NADH + H+ Pseudomonas putida immediate degradation via the NdmCDE complex xanthine + NAD+ + H2O + formaldehyde
-
 ?
1.14.13.128 7-methylxanthine + O2 + NADH + H+ Pseudomonas putida CBB5 immediate degradation via the NdmCDE complex xanthine + NAD+ + H2O + formaldehyde
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.B34 Pseudomonas putida A0A0M3CPH9
-
-
1.14.13.B34 Pseudomonas putida CBB5 A0A0M3CPH9
-
-
1.14.13.128 Pseudomonas putida M1EY73
-
-
1.14.13.128 Pseudomonas putida CBB5 M1EY73
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.B34 recombinant N-terminally MBP-tagged NdmD by affinity chromatography, ion exchange chromatography, and gel filtration, copurification with NdmC and NdmE Pseudomonas putida
1.14.13.128 recombinant His-tagged wild-type and mutant NdmC proteins by nickel affinity chromatography and gel filtration Pseudomonas putida

Source Tissue

EC Number Source Tissue Comment Organism Textmining
1.14.13.B34 cell culture soil bacterium Pseudomonas putida strain CBB5 can use caffeine (1,3,7-trimethylxanthine) as a sole carbon and nitrogen source Pseudomonas putida
-
1.14.13.128 cell culture soil bacterium Pseudomonas putida strain CBB5 can use caffeine (1,3,7-trimethylxanthine) as a sole carbon and nitrogen source Pseudomonas putida
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.B34 additional information the enzyme NdmD shows cytochrome c reductase (ccr, EC 1.1.1.2) activity. NdmD also is the RO reductase that forms a stable ternary complex with NdmC and NdmE (NdmCDE). Since NdmC detaches the N-7 methyl group from methylxanthine derivatives, the NdmCDE complex is responsible for the last N-demethylation step of caffeine to xanthine. But NdmD is also needed by both demethylases NdmA and NdmB for electron transport from NADH to the oxygen activation site, as a demethylase reductase. Therefore, it is expected that transient interaction would exist between them Pseudomonas putida ?
-
-
1.14.13.B34 additional information the enzyme NdmD shows cytochrome c reductase (ccr, EC 1.1.1.2) activity. NdmD also is the RO reductase that forms a stable ternary complex with NdmC and NdmE (NdmCDE). Since NdmC detaches the N-7 methyl group from methylxanthine derivatives, the NdmCDE complex is responsible for the last N-demethylation step of caffeine to xanthine. But NdmD is also needed by both demethylases NdmA and NdmB for electron transport from NADH to the oxygen activation site, as a demethylase reductase. Therefore, it is expected that transient interaction would exist between them Pseudomonas putida CBB5 ?
-
-
1.14.13.128 7-methylxanthine + O2 + NADH + H+ immediate degradation via the NdmCDE complex Pseudomonas putida xanthine + NAD+ + H2O + formaldehyde
-
 ?
1.14.13.128 7-methylxanthine + O2 + NADH + H+ NdmC specifically detaches methyl groups from the N-7 position of methylxanthine derivatives Pseudomonas putida xanthine + NAD+ + H2O + formaldehyde
-
 ?
1.14.13.128 7-methylxanthine + O2 + NADH + H+ immediate degradation via the NdmCDE complex Pseudomonas putida CBB5 xanthine + NAD+ + H2O + formaldehyde
-
 ?
1.14.13.128 7-methylxanthine + O2 + NADH + H+ NdmC specifically detaches methyl groups from the N-7 position of methylxanthine derivatives Pseudomonas putida CBB5 xanthine + NAD+ + H2O + formaldehyde
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.B34 More the enzyme occurs as a Rieske nonheme iron oxygenase (RO)-reductase complex, the NdmCDE heterotrimer. NdmCDE domain architecture analysis, NdmC contains the ligand-binding domain, and the remaining Rieske domain must be nonfunctional because the metal coordinating residues are not conserved. Instead, a potentially functional, unique Rieske domain is located at the N-terminus of NdmD. In addition to the N-terminal Rieske domain, NdmD is composed of a flavin mononucleotide (FMN)-binding domain, an NADH-binding domain, and a C-terminal plant-type ferredoxin domain. NdmE has no discernable function, but exhibits high structural similarity to many glutathione-S-transferases. NdmE might facilitate complex formation by structural alignment Pseudomonas putida
1.14.13.128 More the enzyme occurs as a Rieske nonheme iron oxygenase (RO)-reductase complex, the NdmCDE heterotrimer. NdmCDE domain architecture analysis, NdmC contains the ligand-binding domain, and the remaining Rieske domain must be nonfunctional because the metal coordinating residues are not conserved. Instead, a potentially functional, unique Rieske domain is located at the N-terminus of NdmD. In addition to the N-terminal Rieske domain, NdmD is composed of a flavin mononucleotide (FMN)-binding domain, an NADH-binding domain, and a C-terminal plant-type ferredoxin domain. NdmE has no discernable function, but exhibits high structural similarity to many glutathione-S-transferases. NdmE might facilitate complex formation by structural alignment Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.14.13.B34 NdmD
-
 Pseudomonas putida
1.14.13.B34 Rieske nonheme iron oxygenase reductase
-
 Pseudomonas putida
1.14.13.B34 RO reductase
-
 Pseudomonas putida
1.14.13.128 bacterial N-demethylase
-
 Pseudomonas putida
1.14.13.128 NdmC
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.B34 30
-
 assay at Pseudomonas putida
1.14.13.128 30
-
 assay at Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.B34 7.5
-
 assay at Pseudomonas putida
1.14.13.128 7.5
-
 assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.B34 Ferredoxin
-
 Pseudomonas putida
1.14.13.B34 NADH
-
 Pseudomonas putida
1.14.13.128 NADH
-
 Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.14.13.B34 evolution NdmD is an FNR-type family member and is classified as a type 1A reductase in the two-component system, which is composed of a FMN-binding domain, NADH-binding domain, and C-terminal plant-type ferredoxin domain. There is an additional Rieske domain at the N-terminus of NdmD, which is a unique feature compared with other RO reductases Pseudomonas putida
1.14.13.B34 metabolism Rieske nonheme iron oxygenases (ROs) catalyze the initial oxygenation reaction of aromatic compounds by enantio- and regiospecific reactions. The type of RO in Pseudomonas putida strain CBB5, consists of the monooxygenasesNdmA, NdmB, and NdmC, which specifically detach methyl groups from the N-1, N-3, and N-7 positions of methylxanthine derivatives, respectively. The N-demethylation of caffeine to xanthine occurs via three steps: NdmA and NdmB catalyze the initial two steps of N-demethylation, and the intermediate product, 7-methylxanthine, is further catalyzed to xanthine by an unusual RO-reductase complex, the NdmCDE heterotrimer. Heterohexamerization of NdmA and NdmB under physiological conditions. NdmD is the RO reductase that forms a stable ternary complex with NdmC and NdmE (NdmCDE). Since NdmC detaches the N-7 methyl group from methylxanthine derivatives, the NdmCDE complex is responsible for the last N-demethylation step of caffeine to xanthine. But NdmD is also needed by both NdmA and NdmB for electron transport from NADH to the oxygen activation site. Therefore, it is expected that transient interaction would exist between them. Electron transfer pathway from the ferredoxin domain of NdmD to caffeine in the catalytic site of NdmA. Enzyme complex structure analysis structure-function analysis, overview Pseudomonas putida
1.14.13.B34 additional information analysis of the binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state. Interaction analysis of NdmD with NdmA, B, and C, detailed overview Pseudomonas putida
1.14.13.B34 physiological function some bacteria, such as Pseudomonas putida strain CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes: NdmA, NdmB, NdmC, NdmD, and NdmE Pseudomonas putida
1.14.13.128 metabolism Rieske nonheme iron oxygenases (ROs) catalyze the initial oxygenation reaction of aromatic compounds by enantio- and regiospecific reactions. The type of RO in Pseudomonas putida strain CBB5, consists of NdmA, NdmB, and NdmC, which specifically detach methyl groups from the N-1, N-3, and N-7 positions of methylxanthine derivatives, respectively. A single formaldehyde is produced whenever one N-linked methyl group is detached, indicating that NdmA, NdmB, and NdmC are monooxygenases.The N-demethylation of caffeine to xanthine occurs via three steps; NdmA and NdmB catalyze the initial two steps of N-demethylation, and the intermediate product, 7-methylxanthine, is further catalyzed to xanthine by an unusual RO-reductase complex, the NdmCDE heterotrimer. Heterohexamerization of NdmA and NdmB under physiological conditions. NdmD is the RO reductase that forms a stable ternary complex with NdmC and NdmE (NdmCDE). Since NdmC detaches the N-7 methyl group from methylxanthine derivatives, the NdmCDE complex is responsible for the last N-demethylation step of caffeine to xanthine. But NdmD is also needed by both NdmA and NdmB for electron transport from NADH to the oxygen activation site. Therefore, it is expected that transient interaction would exist between them. Electron transfer pathway from the ferredoxin domain of NdmD to caffeine in the catalytic site of NdmA. Enzyme complex structure analysis structure-function analysis, overview Pseudomonas putida
1.14.13.128 physiological function some bacteria, such as Pseudomonas putida strain CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes: NdmA, NdmB, NdmC, NdmD, and NdmE. Enzyme NdmC specifically detaches methyl groups from the N-7 position of methylxanthine derivatives, NdmC is a monooxygenase Pseudomonas putida