Cloned (Comment) | Organism |
---|---|
gene nrfA, expression of wild-type and mutant enzymes in Escherichia coli strain JCB4083a | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type and mutant Q263E, 10 mg/ml protein, under aerobic conditions by the vapor diffusion hanging drop method using 20% v/v PEG 10000 in 100 mM Na-HEPES, pH 7.5, 20% ethylene glycol as cryoprotectant, X-ray diffraction structure determination and analysis at 1.74 A and 2.04 A resolution, respectively, molecular replacement | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
Q263E | site-directed mutagenesis, the mutation leads to introduction of a negative charge into the vicinity of the active site heme, and the mutant shows reduced activity compared to the wild-type enzyme. The high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics | Escherichia coli | |
0.033 | - |
nitrite | pH 7.0, 25°C, wild-type enzyme with methyl viologen as reductant | Escherichia coli | |
0.413 | - |
nitrite | pH 7.0, 25°C, mutant Q263E with methyl viologen as reductant | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview. An important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand | Escherichia coli | |
Fe3+ | active site heme Fe(III) iron, the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
nitrite + ferrocytochrome c + H+ | Escherichia coli | - |
NH3 + H2O + ferricytochrome c | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0ABK9 | gene nrfA | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain JCB4083a by ammonium sulfate fractionation and anion exchange chromatography | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview. Important function of the Q263-calcium ion pair increasing substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand, active site structures of native and Q263 mutant NrfA enzymes, overview | Escherichia coli | ? | - |
? | |
nitrite + ferrocytochrome c + H+ | - |
Escherichia coli | NH3 + H2O + ferricytochrome c | - |
? | |
nitrite + reduced methyl viologen | - |
Escherichia coli | NH3 + H2O + oxidized methyl viologen | - |
? |
Synonyms | Comment | Organism |
---|---|---|
cytochrome c nitrite reductase | - |
Escherichia coli |
NrfA | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
heme | - |
Escherichia coli |