Information on EC 1.9.6.1 - nitrate reductase (cytochrome)

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.9.6.1
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RECOMMENDED NAME
GeneOntology No.
nitrate reductase (cytochrome)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 ferrocytochrome + 2 H+ + nitrate = 2 ferricytochrome + nitrite
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrate reduction IV (dissimilatory)
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nitrate reduction X (dissimilatory, periplasmic)
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Nitrogen metabolism
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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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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
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Q9RC05
UniProt
Manually annotated by BRENDA team
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Q9RC05
UniProt
Manually annotated by BRENDA team
the genes for the periplasmic nitrate reductase are not part of the bacterial chromosome, but are located on the megaplasmid pHG1 present in the wild-type strain H16
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Manually annotated by BRENDA team
serovar typhimurium
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Manually annotated by BRENDA team
serovar typhimurium
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Shewanella oneidensis MR-1 / ATCC 700550
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
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
2 reduced methyl viologen + 2 H+ + nitrate
2 oxidized methyl viologen + nitrite
show the reaction diagram
nitrate + ferrocytochrome
nitrite + ferricytochrome + H2O
show the reaction diagram
nitrate + reduced acceptor
nitrite + acceptor
show the reaction diagram
nitrate + reduced acceptor
nitrite + oxidized acceptor
show the reaction diagram
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-
-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen
show the reaction diagram
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-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
show the reaction diagram
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen
show the reaction diagram
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-
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?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
show the reaction diagram
nitrite + methyl viologen
nitrate + oxidized methyl viologen
show the reaction diagram
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-
-
-
?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
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
nitrate + reduced acceptor
nitrite + acceptor
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
cytochrome
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cytochrome c
cytochrome c552
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the enzyme is a complex of a 93000 Da polypeptide and a 16000 Da nitrate-oxidizable cytochrome c552, cytochrome c552 contains two c-type heme moieties
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molybdenum cofactor
molybdopterin
[4Fe-4S] cluster
additional information
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enzyme contains no flavin
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron
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contains iron
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
Molybdenum
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
azide
hydrogensulfite
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0.1 mM, complete inhibition
nitrate
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at high substrate concentration, substrate inhibition occurs, mechanism of substrate inhibition in Nap, and kinetics, overview
p-chloromercuribenzoate
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0.3 mM, completely inhibits, can be reversed by cysteine or glutathione
Sulfide
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Na2S at 0.5 mM inactivates completely
Thiocyanate
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competitive
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NapD
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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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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.045 - 0.12
nitrate
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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assay at
7 - 8.5
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same rate between
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 10
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sharp drop of activity below pH 5.5 and above pH 10
7 - 9.5
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pH 7.0: about 70% of maximal activity, pH 9.5: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
37
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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mutant strain (M-6) overproduces the enzyme activity under anaerobic growth conditions
Manually annotated by BRENDA team
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specific activity of the enzyme is higher in intact cells grown with butyrate than succinate as the sole source of carbon
Manually annotated by BRENDA team
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specific activity of the enzyme is higher in intact cells grown with butyrate than succinate as the sole source of carbon
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Cupriavidus necator (strain ATCC 17699 / H16 / DSM 428 / Stanier 337)
Cupriavidus necator (strain ATCC 17699 / H16 / DSM 428 / Stanier 337)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18924
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1 * 93309 + 1 * 18924, calculated from sequence
87000
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1 * 17000 + 1 * 87000, SDS-PAGE
92000
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gel filtration
93000
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1 * 93000 + 1 * 16000, SDS-PAGE
93309
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1 * 93309 + 1 * 18924, calculated from sequence
110000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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1 * 93000 + 1 * 16000, SDS-PAGE
heterodimer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
P33937 and P0ABL3
the pre-NapA leader sequence is both unexpectedly long and, unless two successive proteolysis steps are involved, is cleaved at the unprecedented sequence G-Q-Q
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
X-ray diffraction structure analysis, resolution 2.2 A, PDB ID 2jio, modelling
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purified recombinant NapDNHis/NapA complex, small angle X-ray scattering analysis, modelling
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vapor diffusion method
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sitting-drop vapour-diffusion method, crystals of the oxidized form of this enzyme are obtained using polyethylene glycol 3350 as precipitant. A single crystal diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7°, space group C2, and one heterodimer is present per asymmetric unit
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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10 min, stable
60
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10 min, 10-15% loss of activity
70
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5 min, complete and irreversible loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable to prolonged dialysis
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, about 90% of the activity is lost after 48 h, no activity remains after 4 days
P33937 and P0ABL3
frozen, several months, stable
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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)
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of NapA and NapB
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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P33937 and P0ABL3
cloning of napABC, quantitative PCR-based expression analysis
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expressed in Escherichia coli
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gene nap and nap gene cluster, genetic organization and sequence comparisons
gene nap, expression analysis
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
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structural genes, napA and napB, are cloned, and their nucleotide sequences is determined
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
disruption of nasT leads to a marked decrease in nosZ and nap transcription in cells incubated in the presence of nitrate
in the absence of nitrate, mutated nasS, which encodes the nitrate (NO3-)-sensor of the two-component NasST regulatory system, induces expression of nosZ and periplasmic nitrate reductase nap via nasT. Nitrate addition dissociates the NasS-NasT complex in vitro, suggesting the release of the activator NasT
NarL represses the expression of periplasmic nitrate reductase NapAB under anaerobiosis
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S4C/S24C
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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
additional information
Show AA Sequence (2007 entries)
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