BRENDA - Enzyme Database

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Leadbeater, C.; McIver, L.; Campopiano, D.; Webster, S.; Baxter, R.; Kelly, S.; Price, N.; Lysek, D.; Noble, M.; Chapman, S.; Munro, A.; Biochem. J. 352, 257-266 (2000)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.19.1.1
-
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.19.1.1
molecular modelling indicates that movement of the C-terminal tryptophan (W248) is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. Residues R174 and R184 are located close to the adenosine ribose 2'-phosphate group, and R144 is likely to interact with the nicotinamide ribose 5'-phosphate group
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.19.1.1
R144A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
1.19.1.1
R174A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
1.19.1.1
R184A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.19.1.1
0.0017
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.002
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0039
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0051
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0053
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0099
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0202
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0544
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.19.1.1
Escherichia coli
-
-
-
1.19.1.1
Escherichia coli HMS174
-
-
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.19.1.1
oxidized cytochrome c + NADH + H+
-
737634
Escherichia coli
reduced cytochrome c + NAD+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADH + H+
-
737634
Escherichia coli HMS174
reduced cytochrome c + NAD+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADPH + H+
-
737634
Escherichia coli
reduced cytochrome c + NADP+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADPH + H+
-
737634
Escherichia coli HMS174
reduced cytochrome c + NADP+
-
-
-
r
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.19.1.1
0.23
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.55
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.71
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.84
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
2.2
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
4.03
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
5.1
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
5.65
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.19.1.1
-
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.19.1.1
molecular modelling indicates that movement of the C-terminal tryptophan (W248) is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. Residues R174 and R184 are located close to the adenosine ribose 2'-phosphate group, and R144 is likely to interact with the nicotinamide ribose 5'-phosphate group
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.19.1.1
R144A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
1.19.1.1
R174A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
1.19.1.1
R184A
mutation in the proposed NADPH-binding site, mutant exhibits decreased NADPH-dependent cytochrome c reductase activity and increased Km for NADPH
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.19.1.1
0.0017
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.002
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0039
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0051
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0053
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0099
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0202
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.0544
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.19.1.1
oxidized cytochrome c + NADH + H+
-
737634
Escherichia coli
reduced cytochrome c + NAD+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADH + H+
-
737634
Escherichia coli HMS174
reduced cytochrome c + NAD+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADPH + H+
-
737634
Escherichia coli
reduced cytochrome c + NADP+
-
-
-
r
1.19.1.1
oxidized cytochrome c + NADPH + H+
-
737634
Escherichia coli HMS174
reduced cytochrome c + NADP+
-
-
-
r
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.19.1.1
0.23
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.55
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.71
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
0.84
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
2.2
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
4.03
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
5.1
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
5.65
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.19.1.1
93
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
108
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
270
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
445
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
506
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
715
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
762
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
1448
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.19.1.1
93
-
NADPH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
108
-
NADPH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
270
-
NADH
wild-type, pH 7.5, 30°C
Escherichia coli
1.19.1.1
445
-
NADH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
506
-
NADH
mutant R184A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
715
-
NADH
mutant R174A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
762
-
NADPH
mutant R144A, pH 7.5, 30°C
Escherichia coli
1.19.1.1
1448
-
NADPH
wild-type, pH 7.5, 30°C
Escherichia coli