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Literature summary for 7.1.1.2 extracted from

  • Morina, K.; Schulte, M.; Hubrich, F.; Doerner, K.; Steimle, S.; Stolpe, S.; Friedrich, T.
    Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase (2011), J. Biol. Chem., 286, 34627-34634.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli Escherichia coli

Protein Variants

Protein Variants Comment Organism
E183D site-directed mutagenesis, the mutant shows a 2fold increase in NADH/ferricyanide oxidoreductase activity and a 5fold increase in NADPHoxidase activity compared to the wild-type enzyme Escherichia coli
E183H site-directed mutagenesis, the mutant shows a 2fold increase in NADH/ferricyanide oxidoreductase activity and a 2fold increase in NADPHoxidase activitycompared to the wild-type enzyme Escherichia coli
E183N site-directed mutagenesis, the mutant shows a 2fold increase in NADH/ferricyanide oxidoreductase activity and a 2fold increase in NADPHoxidase activitycompared to the wild-type enzyme Escherichia coli
E183Q site-directed mutagenesis, the mutant shows a similar NADH/ferricyanide oxidoreductase activity and a 2fold increase NADPHoxidase activity compared to the wild-type enzyme Escherichia coli
additional information construction of several variants with mutations at position 183 exhibiting up to 200fold enhanced catalytic efficiency with NADPH Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
Piericidin A
-
Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0057
-
NADH mutant E183H, pH 6.0, 30°C Escherichia coli
0.0058
-
NADH mutant E183D, pH 6.0, 30°C Escherichia coli
0.012
-
NADH mutant E183Q, pH 6.0, 30°C Escherichia coli
0.013
-
NADH wild-type enzyme, pH 6.0, 30°C Escherichia coli
0.014
-
NADH mutant E183N, pH 6.0, 30°C Escherichia coli
0.025
-
NADPH mutant E183H, pH 6.0, 30°C Escherichia coli
0.045
-
NADPH mutant E183Q, pH 6.0, 30°C Escherichia coli
0.39
-
NADPH mutant E183D, pH 6.0, 30°C Escherichia coli
0.48
-
NADPH mutant E183N, pH 6.0, 30°C Escherichia coli
1.87
-
NADPH wild-type enzyme, pH 6.0, 30°C Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane
-
Escherichia coli 16020
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
NADH + ubiquinone + 6 H+[side 1] Escherichia coli
-
NAD+ + ubiquinol + 7 H+[side 2]
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli Escherichia coli

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
NADH/ferricyanide oxidoreductase activity (measured as ferricyanide reduction) and NAD(P)H oxidase activity (measured as oxygen consumption) of cytoplasmic membranes from Escherichia coli recombinantly expressing the enzyme Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
NADH + ubiquinone + 6 H+[side 1]
-
Escherichia coli NAD+ + ubiquinol + 7 H+[side 2]
-
?
NADPH + ubiquinone + 6 H+[side 1] NADPH is a poor substrate of the complex Escherichia coli NADP+ + ubiquinol + 7 H+[side 2]
-
?

Subunits

Subunits Comment Organism
More the enzyme nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide Escherichia coli

Synonyms

Synonyms Comment Organism
complex I
-
Escherichia coli
energy-converting NADPH:ubiquinone oxidoreductase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2 8 NADH mutant E183Q, pH 6.0, 30°C Escherichia coli
3
-
NADPH wild-type enzyme, pH 6.0, 30°C Escherichia coli
10
-
NADPH mutant E183H, pH 6.0, 30°C Escherichia coli
11
-
NADPH mutant E183N, pH 6.0, 30°C Escherichia coli
11
-
NADH mutant E183N, pH 6.0, 30°C Escherichia coli
14
-
NADPH mutant E183Q, pH 6.0, 30°C Escherichia coli
26
-
NADH wild-type enzyme, pH 6.0, 30°C Escherichia coli
34
-
NADPH mutant E183D, pH 6.0, 30°C Escherichia coli
37
-
NADH mutants E183D and E18H, pH 6.0, 30°C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 7 mutant E183H, activity with NADH Escherichia coli
6
-
mutant E183H, activity with NADPH Escherichia coli
6
-
wild-type enzyme, activity with NADH, first peak Escherichia coli
6.5
-
wild-type enzyme, activity with NADPH Escherichia coli
7.5
-
wild-type enzyme, activity with NADH, second higher peak Escherichia coli

pH Range

pH Minimum pH Maximum Comment Organism
5 8 activity range Escherichia coli

Cofactor

Cofactor Comment Organism Structure
FMN
-
Escherichia coli
additional information NADPH is a poor substrate of the complex Escherichia coli
NADH binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex Escherichia coli

General Information

General Information Comment Organism
physiological function the respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.8
-
NADPH wild-type enzyme, pH 6.0, 30°C Escherichia coli
20
-
NADPH mutant E183N, pH 6.0, 30°C Escherichia coli
90
-
NADPH mutant E183D, pH 6.0, 30°C Escherichia coli
320
-
NADPH mutant E183Q, pH 6.0, 30°C Escherichia coli
400
-
NADPH mutant E183H, pH 6.0, 30°C Escherichia coli
800
-
NADH mutant E183N, pH 6.0, 30°C Escherichia coli
2000
-
NADH wild-type enzyme, pH 6.0, 30°C Escherichia coli
2300
-
NADH mutant E183Q, pH 6.0, 30°C Escherichia coli
6400
-
NADH mutant E183D, pH 6.0, 30°C Escherichia coli
6500
-
NADH mutant E183H, pH 6.0, 30°C Escherichia coli