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Literature summary for 1.7.1.3 extracted from
- Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (1998), Arch. Biochem. Biophys., 358, 104-115.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| wild-type and mutants are expressed in Escherichia coli JM109(DE3)pLysS | Neurospora crassa |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | several mutations of recombinant cyt b reductase fragment of nitrate reductase in the region Ser920, Arg921 and Arg932 are created. Conversion from NADPH-specific to virtually NADH-specific cyt b reductase fragment of nitrate reductase | Neurospora crassa |
| R921S | little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity | Neurospora crassa |
| R921T | little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity | Neurospora crassa |
| R932Q | 1/4 wild type NADPH activity is retained, twice as much NADH activity is present as compared to wild type | Neurospora crassa |
| R932S | 1/10 wild type NADPH activity is retained, 2/3 of wild type NADH activity | Neurospora crassa |
| S920D | important for the enzyme's interaction with the pyridine nucleotide substrates. Mutant retains ~2% of the NADPH activity of the wild type while it has an increased NADH activity, ~15% higher. It is concluded that Ser920 is a ligand involved in binding the 2' phosphate of NADPH in the wild type enzyme | Neurospora crassa |
| S920D/R932S | greatest decrease in NADPH activity of all created mutants, shows that Arg932 is a residue interacting with the pyridine nucleotide coenzyme electron donors and that Ser920 and Arg932 have effects on substrate binding and catalytic activity. Both residues may be ligands to the 2' phosphate of NADPH in the wild type cyt b reductase fragment of nitrate reductase | Neurospora crassa |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.013 | - |
NADPH | wild type | Neurospora crassa |  |
| 0.25 | - |
NADPH | S920D mutant | Neurospora crassa | |
| 0.25 | - |
NADPH | R932S mutant | Neurospora crassa | |
| 0.4 | - |
NADH | S920D mutant | Neurospora crassa | |
| 2.3 | - |
NADH | S920D/R932S double mutant | Neurospora crassa | |
| 2.6 | - |
NADH | R932S mutant | Neurospora crassa | |
| 3 | - |
NADH | wild type | Neurospora crassa | |
| 4.2 | - |
NADPH | S920D/R932S double mutant | Neurospora crassa | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Molybdenum | - |
Neurospora crassa | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| nitrate + NADPH | Neurospora crassa | - |
nitrite + NADP+ + H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Neurospora crassa | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| metal-chelate affinity chromatography for his-tagged proteins, ammonium sulfate fractionation, affinity chromatography | Neurospora crassa |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| nitrite + NADP+ + H2O = nitrate + NADPH + H+ | multicenter redox enzyme. Ser920, Arg921 and Arg932 are suggested to be the key enzymes to investigate for a role in determining pyridine nucleotide specificity. Arg932 may be playing a role in binding the adenine ring of NADPH | Neurospora crassa |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C, 25 mM Mops, 0.1 mM EDTA, pH 7.2 | Neurospora crassa |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| nitrate + NADH + H+ | - |
Neurospora crassa | nitrite + NAD+ + H2O | - |
r | |
| nitrate + NADPH | - |
Neurospora crassa | nitrite + NADP+ + H2O | - |
? | |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
- |
Neurospora crassa |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | - |
less than 50% loss of activity in 60 min for wild type and S920D mutant | Neurospora crassa |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 18 | - |
NADPH | R920D/R932S double mutant | Neurospora crassa | |
| 180 | - |
NADPH | S920D mutant | Neurospora crassa | |
| 275 | - |
NADH | R932S mutant | Neurospora crassa | |
| 290 | - |
NADH | wild type | Neurospora crassa | |
| 450 | - |
NADPH | R932S mutant | Neurospora crassa | |
| 590 | - |
NADH | R920D/R932S double mutant | Neurospora crassa | |
| 1400 | - |
NADPH | wild type | Neurospora crassa | |
| 1700 | - |
NADH | S920D mutant | Neurospora crassa | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6 | - |
wild type after his-tag is removed | Neurospora crassa |
| 7 | - |
his-tagged wild type | Neurospora crassa |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | - |
Neurospora crassa | |
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