Cloned (Comment) | Organism |
---|---|
expression of wild-type, H327A and H369A mutant enzyme in Pseudomonas putida | Pseudomonas aeruginosa |
Crystallization (Comment) | Organism |
---|---|
crystals of the H327A mutant are obtained by vapor diffusion technique by mixing in a 1:1 ratio the protein and a reservoir solution containing 4.0% polyethylene glycol 5000 monomethyl ether, 0.1 M sodium acetate, pH 5.5. The space group is 4(3)22 with cell dimensions 70.5 x 70.5 x 281 A. Crystals of the H369A mutant are obtained by mixing in a 1.1 ratio the protein and a reservoir solution containing 11.5% polyethylene glycol 6000, 0.2 M imidazole/malate, pH 6.5. The space group is P4(1)2(1)2 with cell dimensions 94.7 x 94.7 x 159.9 A | Pseudomonas aeruginosa |
H327A mutant enzymes: vapour diffusion technique, mixing of the enzyme and a reservoir solution containing 4% polyethylene glycol 5000 monomethyl ether, 100 mM sodium acetate pH 5.5 in a 1/1 ratio, H369A mutant enzyme: 11.5% polyethylene glycol 6000, 200 mM imidazole/malate pH 6.5, x-ray structure of both mutants | Pseudomonas aeruginosa |
Protein Variants | Comment | Organism |
---|---|---|
H327A | reduction of nitrite is severely compromised | Pseudomonas aeruginosa |
H327A | mutant protein has no nitrite reductase activity but maintains the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form | Pseudomonas aeruginosa |
H369A | reduction of nitrite is severely compromised | Pseudomonas aeruginosa |
H369A | mutant protein has no nitrite reductase activity but maintains the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form | Pseudomonas aeruginosa |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0018 | - |
ferricytochrome c551 | recombinant H327A mutant enzyme, O2 reduction | Pseudomonas aeruginosa | |
0.0018 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, mutant enzyme H327A | Pseudomonas aeruginosa | |
0.002 | - |
ferricytochrome c551 | recombinant wild-type enzyme, O2 reduction | Pseudomonas aeruginosa | |
0.002 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, wild-type enzyme | Pseudomonas aeruginosa | |
0.006 | - |
NO2- | recombinant wild-type enzyme | Pseudomonas aeruginosa | |
0.006 | - |
nitrite | pH 6.2, 25°C, wild-type enzyme | Pseudomonas aeruginosa | |
0.0075 | - |
ferricytochrome c551 | recombinant H369A mutant enzyme, O2 reduction | Pseudomonas aeruginosa | |
0.0075 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, mutant enzyme H369A | Pseudomonas aeruginosa |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | P24474 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type, H327A and H369A mutant enzyme | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ferrocytochrome c-551 + O2 | - |
Pseudomonas aeruginosa | ferricytochrome c-551 + H2O | - |
? | |
nitric oxide + H2O + ferricytochrome c551 | - |
Pseudomonas aeruginosa | nitrite + ferrocytochrome c551 + H+ | - |
r | |
nitrite + electron donor | - |
Pseudomonas aeruginosa | NO + oxidized electron donor + H2O | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.025 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, wild-type enzyme | Pseudomonas aeruginosa | |
0.032 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, mutant enzyme H369A | Pseudomonas aeruginosa | |
0.043 | - |
Ferrocytochrome c-551 | pH 6.2, 25°C, mutant enzyme H327A | Pseudomonas aeruginosa | |
0.08 | - |
NO2- | recombinant H327A and H369a mutant enzymes | Pseudomonas aeruginosa | |
0.08 | - |
nitrite | pH 6.2, 25°C, mutant enzyme H327A | Pseudomonas aeruginosa | |
0.08 | - |
nitrite | pH 6.2, 25°C, mutant enzyme H369A | Pseudomonas aeruginosa | |
8 | - |
NO2- | recombinant wild-type enzyme | Pseudomonas aeruginosa | |
8 | - |
nitrite | pH 6.2, 25°C, wild-type enzyme | Pseudomonas aeruginosa |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
Heme d1 | reduction of the substrate occurs at the d1-heme site | Pseudomonas aeruginosa |