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Literature summary extracted from

  • Dow, J.M.; Grahl, S.; Ward, R.; Evans, R.; Byron, O.; Norman, D.G.; Palmer, T.; Sargent, F.
    Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone (2014), FEBS J., 281, 246-260 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.9.6.1 NapD a small (9.3 kDa) cytoplasmic protein that is essential for Nap activity, role for NapD in the insertion of the molybdenum cofactor. The NapD cysteine residues (C8 and C32) are not conserved and a cysteine-free variant of NapD complements a DELTAnapD strain for restoration of NapA activity. A NapD C8S/C32A variant remains attached to the NapA signal peptide. Copurification of recombinant NapA complexed with N-terminally His-tagged NapD activator by nickel afinity chromatography Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.9.6.1 gene napA, enzyme NapA is encoded, along with its periplasmic di-heme c-type cytochrome redox partner NapB, in the seven gene nap operon, coexpression of NapA with His-tagged NapD activator in Escherichia coli strains MC4100 and BL21(DE3), recombinant expression of MTSL-labelled MalE-NapASP fusion mutant S4C/S24C in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain LCB2048 Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.9.6.1 purified recombinant NapDNHis/NapA complex, small angle X-ray scattering analysis, modelling Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
1.9.6.1 S4C/S24C site-directed mutagenesis, native, NapD results in a loss of some of the spin labels from the NapA signal peptide possibly due to the surface-exposed native cysteine residues of NapD. The NapD cysteine residues (C8 and C32) are not conserved and a cysteine-free variant of NapD complements a DELTAnapD strain for restoration of NapA activity. A NapD C8S/C32A variant remains attached to the NapA signal peptide Escherichia coli

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.9.6.1 periplasm NapA is exported to the periplasm in a folded form by the twin-arginine protein transport (Tat) pathway. NapA is subject to Tat proofreading prior to export by the Tat pathway Escherichia coli
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.9.6.1 Fe2+ in the cofactor heme and the [4Fe-4S] cluster Escherichia coli
1.9.6.1 Molybdenum in the molybdenum cofactor. Role for NapD in the insertion of the molybdenum cofactor Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.9.6.1 2 ferrocytochrome + 2 H+ + nitrate Escherichia coli
-
 2 ferricytochrome + nitrite
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.9.6.1 Escherichia coli P33937
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.9.6.1 copurification of recombinant NapA complexed with N-terminally His-tagged NapD activator by nickel afinity chromatography from Escherichia coli strains MC4100 and BL21(DE3) Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.9.6.1 2 ferrocytochrome + 2 H+ + nitrate
-
 Escherichia coli 2 ferricytochrome + nitrite
-
 ?

Subunits

EC Number Subunits Comment Organism
1.9.6.1 More PELDOR analysis of recombinant MTSL-labelled MalE-NapASP fusion mutant S4C/S24C alone or in complex with NapD, comparison of bound, NMR-derived NapASP helix from PDB ID 2PQ4 versus free generated helix, positions of the spin labels in the two conformations of the signal peptide, overview Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
1.9.6.1 NapA
-
 Escherichia coli
1.9.6.1 periplasmic nitrate reductase
-
 Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.9.6.1 cytochrome c di-heme cytochrome c redox partner, NapB Escherichia coli
1.9.6.1 molybdenum cofactor
-
 Escherichia coli
1.9.6.1 [4Fe-4S] cluster
-
 Escherichia coli

General Information

EC Number General Information Comment Organism
1.9.6.1 additional information NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twinarginine signal peptide of NapA. NapA is structured in its unbound form. The NapA signal peptide undergoes conformational rearrangement upon interaction with NapD. NapA is at least partially folded when bound by its NapD partner. The NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor Escherichia coli
1.9.6.1 physiological function Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the involved periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway Escherichia coli