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Literature summary extracted from

  • Shiraishi, N.; Croy, C.; Kaur, J.; Campbell, W.H.
    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.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.7.1.3 wild-type and mutants are expressed in Escherichia coli JM109(DE3)pLysS Neurospora crassa

Protein Variants

EC Number Protein Variants Comment Organism
1.7.1.3 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
1.7.1.3 R921S little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity Neurospora crassa
1.7.1.3 R921T little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity Neurospora crassa
1.7.1.3 R932Q 1/4 wild type NADPH activity is retained, twice as much NADH activity is present as compared to wild type Neurospora crassa
1.7.1.3 R932S 1/10 wild type NADPH activity is retained, 2/3 of wild type NADH activity Neurospora crassa
1.7.1.3 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
1.7.1.3 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.7.1.3 0.013
-
 NADPH wild type Neurospora crassa
1.7.1.3 0.25
-
 NADPH S920D mutant Neurospora crassa
1.7.1.3 0.25
-
 NADPH R932S mutant Neurospora crassa
1.7.1.3 0.4
-
 NADH S920D mutant Neurospora crassa
1.7.1.3 2.3
-
 NADH S920D/R932S double mutant Neurospora crassa
1.7.1.3 2.6
-
 NADH R932S mutant Neurospora crassa
1.7.1.3 3
-
 NADH wild type Neurospora crassa
1.7.1.3 4.2
-
 NADPH S920D/R932S double mutant Neurospora crassa

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.7.1.3 Molybdenum
-
 Neurospora crassa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.7.1.3 nitrate + NADPH Neurospora crassa
-
 nitrite + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.7.1.3 Neurospora crassa
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.7.1.3 metal-chelate affinity chromatography for his-tagged proteins, ammonium sulfate fractionation, affinity chromatography Neurospora crassa

Reaction

EC Number Reaction Comment Organism Reaction ID
1.7.1.3 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
a

Storage Stability

EC Number Storage Stability Organism
1.7.1.3 -80°C, 25 mM Mops, 0.1 mM EDTA, pH 7.2 Neurospora crassa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.7.1.3 nitrate + NADH + H+
-
 Neurospora crassa nitrite + NAD+ + H2O
-
 r
1.7.1.3 nitrate + NADPH
-
 Neurospora crassa nitrite + NADP+ + H2O
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.7.1.3 25
-
-
 Neurospora crassa

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.7.1.3 40
-
 less than 50% loss of activity in 60 min for wild type and S920D mutant Neurospora crassa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.7.1.3 18
-
 NADPH R920D/R932S double mutant Neurospora crassa
1.7.1.3 180
-
 NADPH S920D mutant Neurospora crassa
1.7.1.3 275
-
 NADH R932S mutant Neurospora crassa
1.7.1.3 290
-
 NADH wild type Neurospora crassa
1.7.1.3 450
-
 NADPH R932S mutant Neurospora crassa
1.7.1.3 590
-
 NADH R920D/R932S double mutant Neurospora crassa
1.7.1.3 1400
-
 NADPH wild type Neurospora crassa
1.7.1.3 1700
-
 NADH S920D mutant Neurospora crassa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.7.1.3 6
-
 wild type after his-tag is removed Neurospora crassa
1.7.1.3 7
-
 his-tagged wild type Neurospora crassa

Cofactor

EC Number Cofactor Comment Organism Structure
1.7.1.3 FAD
-
 Neurospora crassa