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Information on EC 1.14.13.B34 - N-demethylase reductase
for references in articles please use BRENDA:EC1.14.13.B34preliminary BRENDA-supplied EC number
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EC Tree
1.14.13.B34 N-demethylase reductaseSpecify your search results
The enzyme appears in viruses and cellular organisms
Synonyms
ro reductase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
NdmD
Rieske nonheme iron oxygenase reductase
RO reductase
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SYSTEMATIC NAME
IUBMB Comments
N-demethylase oxidoreductase
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
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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
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additional information
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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
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additional information
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enzyme NdmD shows broad substrate specificity with highest catalytic efficiency towards cytochrome c. The enzyme NdmD shows cytochrome c reductase (ccr, EC 1.1.1.2) activity towards three different substrates cytochrome c, DCPIP, and potassium ferricyanide,, kinetics overviews. NdmD also assists as an oxidoreductase the demethylase activity of NdmA, NdmB, and NdmC, overview. NdmD is the reductase component of the different methylxanthine demethylases. NdmD does not catalyze caffeine demethylation but only acts as a reductase
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
Mg2+ and Mn2+ do not cause any significant change in enzyme activity
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
23.88
purified recombinant enzyme, pH 8.0, 25°C, substrate cytochrome c
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 10
40% of maximal activity at pH 6.0, 60% at pH 10.0
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15 - 45
activity range, a drastic drop in activity is observed above 40°C, 16% of maximal activity at 45°C
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isolated from coffee plantation soil in Ooty, India
UniProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
soil bacterium Pseudomonas putida strain CBB5 can use caffeine (1,3,7-trimethylxanthine) as a sole carbon and nitrogen source
brenda
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soil bacterium Pseudomonas putida strain CBB5 can use caffeine (1,3,7-trimethylxanthine) as a sole carbon and nitrogen source
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brenda
strain NCIM 5235 can utilize caffeine as sole source of carbon and nitrogen for growth
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
65% loss in reductase activity of NdmD can be attributed to the fractional loss of flavin by disruption of the flavin-binding environment of FMN-binding pocket. Deletion of N-terminal Rieske domain does not disrupt flavin binding in the constructs DELTA114NdmD and DELTA250NdmD
metabolism
physiological function
additional information
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
evolution
the N-terminal Rieske domain found in NdmD is a product of domain shuffling between NdmC and NdmD during evolution and is not required for its reductase activity
evolution
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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
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metabolism
based on the sequence of a genomic fragment, caffeine demethylation enzyme system found in Pseudomonas sp. is predicted to consist of a two two-component Rieske monooxygenases namely NdmA and NdmB specific towards methyl groups at 1 and 3 positions in xanthine ring respectively and one kind of three-component Rieske monooxygenase system comprising a monooxygenase NdmC specific towards 7-methylxanthine, a reductase component NdmD and a structural protein NdmE. NdmD also acts as the reductase component for NdmA and NdmB. The Rieske domain present in NdmD serves to function as an electron transfer domain during catalysis by NdmC as it lacks its own Rieske domain. NdmC forms a large multi-subunit complex comprising 2 monomeric units of each NdmC, NdmD, and NdmE and follows the typical electron flow pattern of Rieske oxygenases. The N-terminal Rieske domain found in NdmD is a product of domain shuffling between NdmC and NdmD during evolution and is not required for its reductase activity
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
metabolism
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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
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physiological function
Pseudomonas sp. NCIM 5235 is a caffeine-degrading bacterial strain that metabolizes caffeine by sequential demethylation using methylxanthine demethylases, including 7-methylxanthine demethylase NdmC. These enzymes belong to the class of two-component Rieske oxygenases and require an oxidoreductase, NdmD, for efficient catalysis. Three oxygenases (NdmA, NdmB, and NdmC) specific towards methyl groups at 1, 3, and 7 positions in xanthine ring share a common reductase component, NdmD, analysis of NdmD parameters and function, overview. NdmD acts as the reductase component for NdmA, NdmB, and NdmC. The Rieske domain present in NdmD serves to function as an electron transfer domain during catalysis by NdmC as it lacks its own Rieske domain
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
physiological function
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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
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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
additional information
NdmD in Pseudomonas sp. has a unique domain fusion in its N-terminal that is not observed in any other Rieske oxygenase reductases reported so far. The N-terminal Rieske domain found in NdmD is a product of domain shuffling between NdmC and NdmD during evolution and is not required for its reductase activity
additional information
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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
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 65000, about, recombinant His-tagged NdmD, SDS-PAGE
additional information
additional information
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
additional information
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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
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additional information
NdmC forms a large multi-subunit complex comprising 2 monomeric units of each NdmC, NdmD, and NdmE and follows the typical electron flow pattern of Rieske oxygenases. The Rieske domain present in NdmD serves to function as an electron transfer domain during catalysis by NdmC as it lacks its own Rieske domain. Enzyme domain structure, overview
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
to determine the role of N-terminal Rieske domain in NdmD reductase activity, two deletion constructs DELTA114NdmD and DELTA250NdmD are made. Cytochrome c reductase (ccr) activity of the NdmD constructs and demethylase activity of NdmA in the presence of NdmD constructs show that there is no significant difference in the catalytic activity of NdmD upon deletion of its N-terminal Rieske domain
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged NdmD 3.63fold from Escherichia coli strain BL21 by nickel affinity chromatography
recombinant N-terminally MBP-tagged NdmD by affinity chromatography, ion exchange chromatography, and gel filtration, copurification with NdmC and NdmE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ndmD, genetic organization of caffeine-degrading genes and its isolation and of the ndmD gene, recombinant expression of His-tagged wild-type and mutant NdmDs in Escherichia coli strain BL21
gene ndmD, recombinant overexpression of N-terminally MBP-tagged NdmD, coexpression with NdmC and NdmE
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Retnadhas, S.; Gummadi, S.N.
Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM 5235
Appl. Microbiol. Biotechnol.
102
7913-7926
2018
Pseudomonas sp. NCIM 5235 (A0A2U9IY48)
brenda
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
J. Mol. Biol.
431
3647-3661
2019
Pseudomonas putida (A0A0M3CPH9), Pseudomonas putida CBB5 (A0A0M3CPH9)
brenda
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Release 2023.1 (January 2023)
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