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Literature summary for 1.14.13.128 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)

Cloned (Comment) Organism
gene ndmC, recombinant overexpression of His-tagged wild-type and mutant NdmC proteins, coexpression with NdmD and NdmE Pseudomonas putida

Protein Variants

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Pseudomonas putida M1EY73
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Pseudomonas putida CBB5 M1EY73
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-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant NdmC proteins by nickel affinity chromatography and gel filtration Pseudomonas putida

Source Tissue

Source Tissue Comment Organism Textmining
cell culture soil bacterium Pseudomonas putida strain CBB5 can use caffeine (1,3,7-trimethylxanthine) as a sole carbon and nitrogen source Pseudomonas putida
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Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7-methylxanthine + O2 + NADH + H+ immediate degradation via the NdmCDE complex Pseudomonas putida xanthine + NAD+ + H2O + formaldehyde
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?
7-methylxanthine + O2 + NADH + H+ NdmC specifically detaches methyl groups from the N-7 position of methylxanthine derivatives Pseudomonas putida xanthine + NAD+ + H2O + formaldehyde
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?
7-methylxanthine + O2 + NADH + H+ immediate degradation via the NdmCDE complex Pseudomonas putida CBB5 xanthine + NAD+ + H2O + formaldehyde
-
?
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

Subunits Comment Organism
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

Synonyms Comment Organism
bacterial N-demethylase
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Pseudomonas putida
NdmC
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Pseudomonas putida

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
30
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assay at Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Pseudomonas putida

Cofactor

Cofactor Comment Organism Structure
NADH
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Pseudomonas putida

General Information

General Information Comment Organism
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
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