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Information on EC 1.9.6.1 - nitrate reductase (cytochrome) and Organism(s) Escherichia coli and UniProt Accession P33937

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Escherichia coli
UNIPROT: P33937 not found.
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
periplasmic nitrate reductase, periplasmic nitrate reductases, napedabc, napabc, nap-alpha, nap-beta, nap enzyme, benzyl viologen-nitrate reductase, napdaghb, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
periplasmic nitrate reductase
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benzyl viologen-nitrate reductase
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periplasmic nitrate reductases
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reductase, nitrate (cytochrome)
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respiratory nitrate reductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 ferrocytochrome + 2 H+ + nitrate = 2 ferricytochrome + nitrite
show the reaction diagram
sulfur-shift mechanism catalytic mechanism, detailed overview. The mechanism is defined by a change in the Mo ion coordination, which involves a first-to-second shell displacement (shift) of the sulfur from the Cys, resulting in a free coordination position that is used by the enzyme to bind the substrate with a low energy cost, molybdenum coordinates an oxygen atom from the substrate, an oxygen atom from the substrate is transferred to the Mo ion, and later released as a water molecule. The reaction requires two electrons, which are provided by external reducing species, and two protons that are obtained from the solvent either directly or indirectly mediated by residues from the enzyme catalytic pocket
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
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oxidation
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reduction
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
ferrocytochrome:nitrate oxidoreductase
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CAS REGISTRY NUMBER
COMMENTARY hide
9029-42-9
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 ferrocytochrome + 2 H+ + nitrate
2 ferricytochrome + nitrite
show the reaction diagram
nitrate + ferrocytochrome
nitrite + ferricytochrome + H2O
show the reaction diagram
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the periplasmic cytochrome c-linked nitrate reductase is encoded by the napFDAGHBC operon. The napF operon apparently encodes a low-substrate-induced reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a high-substrate-induced operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Nitrite, the end product of each enzyme, has only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, is essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2 ferrocytochrome + 2 H+ + nitrate
2 ferricytochrome + nitrite
show the reaction diagram
nitrate + ferrocytochrome
nitrite + ferricytochrome + H2O
show the reaction diagram
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the periplasmic cytochrome c-linked nitrate reductase is encoded by the napFDAGHBC operon. The napF operon apparently encodes a low-substrate-induced reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a high-substrate-induced operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Nitrite, the end product of each enzyme, has only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, is essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c
molybdenum cofactor
[4Fe-4S] cluster
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
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NapA contains a molybdo-bis(molybdopterin guanine dinucleotide) cofactor
heme
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the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB)
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mo(VI)
coordinates a cysteine and a sulfido residue
Molybdenum
in the molybdenum cofactor. Role for NapD in the insertion of the molybdenum cofactor
Fe
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NapA contains a [4Fe-4S] cluster
Mo5+
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NapA contains a molybdo-bis(molybdopterin guanine dinucleotide) cofactor. The molybdenum ion coordination sphere of NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. In NapA or NapAB, the Mo5+ state can not be further reduced to Mo4+. A catalytic cycle for NapA is proposed in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate
Mo6+
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NapA contains a molybdo-bis(molybdopterin guanine dinucleotide) cofactor. The molybdenum ion coordination sphere of NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. In NapA or NapAB, the Mo5+ state can not be further reduced to Mo4+. A catalytic cycle for NapA is proposed in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the prokaryotic nitrate reductases can be subgrouped as respiratory nitrate reductases (Nar), assimilatory nitrate reductases (Nas), and periplasmic nitrate reductases (Nap). Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh), both belonging to the DMSO reductase family, subfamily I, have a very similar structure, but very different activities. The show key differences that tune them for completely different functions in living cells. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. Detailed comparison, overview. A key difference between the catalytic mechanisms of Nap and FdH is the fact that only Mo is used to reduce nitrate but in Fdhs both Mo and W are catalytically competent to oxidize formate to carbon dioxide
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
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17000
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1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
90000
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1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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NapF plays a role in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant NapDNHis/NapA complex, small angle X-ray scattering analysis, modelling
vapor diffusion method
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
copurification of recombinant NapA complexed with N-terminally His-tagged NapD activator by nickel afinity chromatography from Escherichia coli strains MC4100 and BL21(DE3)
of NapA and NapB
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene nap and nap gene cluster, genetic organization and sequence comparisons
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Jepson, B.J.; Mohan, S.; Clarke, T.A.; Gates, A.J.; Cole, J.A.; Butler, C.S.; Butt, J.N.; Hemmings, A.M.; Richardson, D.J.
Spectropotentiometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli
J. Biol. Chem.
282
6425-6437
2006
Escherichia coli
Manually annotated by BRENDA team
Nilavongse, A.; Brondijk, T.H.; Overton, T.W.; Richardson, D.J.; Leach, E.R.; Cole, J.A.
The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA
Microbiology
152
3227-3237
2006
Escherichia coli
Manually annotated by BRENDA team
Berks, B.C.; Ferguson, S.J.; Moir, J.W.; Richardson, D.J.
Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions.
Biochim. Biophys. Acta
1232
97-173
1995
Cupriavidus necator, Escherichia coli, Paracoccus denitrificans, Paracoccus pantotrophus
Manually annotated by BRENDA team
Wang, H.; Tseng, C.P.; Gunsalus, R.P.
The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite
J. Bacteriol.
181
5303-5308
1999
Escherichia coli
Manually annotated by BRENDA team
Gonzalez, P.; Rivas, M.; Mota, C.; Brondino, C.; Moura, I.; Moura, J.
Periplasmic nitrate reductases and formate dehydrogenases biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role
Coord. Chem. Rev.
257
315-331
2013
Anaeromyxobacter dehalogenans (Q2IPE7), Bradyrhizobium japonicum, Campylobacter jejuni subsp. jejuni (Q9PPD9), Campylobacter jejuni subsp. jejuni ATCC 700819 (Q9PPD9), Cereibacter sphaeroides (Q53176), Cupriavidus necator (P39185), Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 (P39185), Desulfitobacterium hafniense (A0A098B5Y5), Desulfovibrio desulfuricans (P81186), Escherichia coli (P33937), Paracoccus denitrificans (A1BB88), Paracoccus pantotrophus (Q56350), Paracoccus pantotrophus GB17 (Q56350), Pseudomonas sp. (Q9RC05), Pseudomonas sp. G-179 (Q9RC05), Shewanella gelidimarina (E2F391), Shewanella oneidensis, Wolinella succinogenes
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Manually annotated by BRENDA team
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
FEBS J.
281
246-260
2014
Escherichia coli (P33937), Escherichia coli
Manually annotated by BRENDA team