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Literature summary for 1.7.1.3 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)

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