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Literature summary for 1.7.1.3 extracted from

  • Ringel, P.; Krausze, J.; Van Heuvel, J.; Curth, U.; Pierik, A.; Herzog, S.; Mendel, R.; Kruse, T.
    Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa (2013), J. Biol. Chem., 288, 14657-14671 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
sequence comparisons, recombinant enzyme expression of wild-type and mutant enzymes in Escherichia coli strains TP1000, RK5206, and RK5204 Neurospora crassa

Protein Variants

Protein Variants Comment Organism
G811V site-directed mutagenesis, an FAD-binding mutant Neurospora crassa
H654A/H677A site-directed mutagenesis, CD spectroscopy shows no negative effects of the introduced mutations on protein secondary structure in comparison to the wild-type protein, but the mutant contains no heme, while the FAD binding ability is not significantly disturbed Neurospora crassa
R778E site-directed mutagenesis, an FAD-binding mutant. The mutant binds essentially the same amount of Moco as does the wild type protein Neurospora crassa
Y780A site-directed mutagenesis, an FAD-binding mutant Neurospora crassa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics, holo-enzyme Neurospora crassa
0.25
-
nitrate recombinant holo-enzyme, pH 7.5, temperature not specified in the publication Neurospora crassa

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ heme Neurospora crassa
Mo5+ in the active site-bound molybdenum cofactor Neurospora crassa

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
additional information
-
gel filtration and analytical ultracentrifugation Neurospora crassa

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Neurospora crassa
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme from Escherichia coli strains TP1000, RK5206, and RK5204 by a two-step affinity purification Neurospora crassa

Specific Activity [micromol/min/mg]

Specific Activity Minimum [Āµmol/min/mg] Specific Activity Maximum [Āµmol/min/mg] Comment Organism
4.32
-
purified recombinant holo-enzyme, substrate nitrate, pH 7.5, temperature not specified in the publication Neurospora crassa

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information NADPH-dependent cytochrome c reducing activity by the holo-enzyme is determined with FAD and NADPH spectroscopically at 550 nm and 340 nm. Apo-nitrate reductase has a marginally lower, about 10% reduced cytochrome c reducing activity, which correlates to its 15% reduced heme content Neurospora crassa ?
-
?
nitrate + NADPH + H+
-
Neurospora crassa nitrite + NADP+ + H2O
-
?

Subunits

Subunits Comment Organism
homodimer holo-enzyme, determination with SDS-PAGE, gel filtration and analytical ultracentrifugation. The enzyme has a largely hydrophobic dimer interface Neurospora crassa
More the apo-enzyme dissociates completely into monomers, the ratio between monomeric and dimeric apo-NR does not change significantly upon a 20fold dilution. Active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization, molybdenum cofactor-dependent enzyme maturation, overview. Enzyme domain structure, overview Neurospora crassa

Synonyms

Synonyms Comment Organism
nitrate reductase [NADPH]
-
Neurospora crassa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
assay at Neurospora crassa

Cofactor

Cofactor Comment Organism Structure
cytochrome c
-
Neurospora crassa
FAD the enzyme contains a a ferredoxin reductase-type FAD binding domain Neurospora crassa
heme enzyme heme binding is independent from Moco/MPT binding Neurospora crassa
molybdenum cofactor i.e Moco/MPT, binding of molybdenum cofactor to apo-nitrate reductase is independent from other prosthetic groups, molybdenum cofactor-dependent enzyme maturation, overview. Reconstitution of Moco-free nitrate reductase with various amounts of purified Moco carrier protein Neurospora crassa
molybdopterin i.e Moco/MPT, binding of molybdenum cofactor to apo-nitrate reductase is independent from other prosthetic groups, molybdenum cofactor-dependent enzyme maturation, overview. Reconstitution of Moco-free nitrate reductase with various amounts of purified Moco carrier protein Neurospora crassa
NADPH dependent on Neurospora crassa

General Information

General Information Comment Organism
additional information active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization, molybdenum cofactor-dependent enzyme maturation, overview Neurospora crassa