Information on EC 1.7.99.4 - nitrate reductase

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

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EC NUMBER
COMMENTARY hide
1.7.99.4
-
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RECOMMENDED NAME
GeneOntology No.
nitrate reductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrite + acceptor = nitrate + reduced acceptor
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
-
-
Nitrogen metabolism
-
-
nitrate assimilation
-
-
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SYSTEMATIC NAME
IUBMB Comments
nitrite:acceptor oxidoreductase
The Pseudomonas enzyme is a cytochrome, but the enzyme from Micrococcus halodenitrificans is an iron protein containing molybdenum. Reduced benzyl viologen and other dyes bring about the reduction of nitrate.
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CAS REGISTRY NUMBER
COMMENTARY hide
37256-45-4
-
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cultivar Smooth Cayenne
UniProt
Manually annotated by BRENDA team
strain Sp245, NAP is expressed under oxic and anoxic conditions
-
-
Manually annotated by BRENDA team
strain Sp245, NAP is expressed under oxic and anoxic conditions
-
-
Manually annotated by BRENDA team
strain S244
-
-
Manually annotated by BRENDA team
strain S244
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain ATCC 27774
-
-
Manually annotated by BRENDA team
strain IFO 12935
-
-
Manually annotated by BRENDA team
strain IFO 12935
-
-
Manually annotated by BRENDA team
strain A1004a, 5-aminolaevulinic acid auxotroph
-
-
Manually annotated by BRENDA team
strain EMG 29
-
-
Manually annotated by BRENDA team
strain EMG-2
-
-
Manually annotated by BRENDA team
strain RK7
-
-
Manually annotated by BRENDA team
strain X5119
-
-
Manually annotated by BRENDA team
Fusarium oxysporum 11dn1
-
-
Manually annotated by BRENDA team
NCA strain 2184, identical with ATCC 12016
-
-
Manually annotated by BRENDA team
extreme halophile
-
-
Manually annotated by BRENDA team
I3R9M9: alpha-subunit (narG), I3R9M8: beta subunit (narH)
I3R9M9 and I3R9M8
SwissProt
Manually annotated by BRENDA team
halophilic archaeon
-
-
Manually annotated by BRENDA team
strain ATCC 13511, moderate halophile, nitrate reductase A
-
-
Manually annotated by BRENDA team
strain AGJ1-3
-
-
Manually annotated by BRENDA team
strain AGJ1-3
-
-
Manually annotated by BRENDA team
strain MS-1
Swissprot
Manually annotated by BRENDA team
strain MS-1
Swissprot
Manually annotated by BRENDA team
strain NCIB 8944
-
-
Manually annotated by BRENDA team
strain LMD82.5, expression of two catalytically distinct forms: a membrane-bound form under anaerobic growth conditions and a periplasmic form mainly under aerobic growth conditions
-
-
Manually annotated by BRENDA team
strain M-6
-
-
Manually annotated by BRENDA team
strain ATCC 13867
-
-
Manually annotated by BRENDA team
strain YT101
-
-
Manually annotated by BRENDA team
strain DSM158, in vitro enzyme activity after different growth conditions
-
-
Manually annotated by BRENDA team
strain RF-1, diazotrophically growing cells
-
-
Manually annotated by BRENDA team
strain RF-1, diazotrophically growing cells
-
-
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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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
chlorate + reduced acceptor
?
show the reaction diagram
chlorate + reduced acceptor
chlorite + acceptor
show the reaction diagram
dithionite + methyl viologen
?
show the reaction diagram
nitrate + reduced acceptor
?
show the reaction diagram
nitrate + reduced acceptor
nitrite + acceptor
show the reaction diagram
nitrate + reduced acceptor
nitrite + acceptor + H2O
show the reaction diagram
-
Wolinella succinogenes transfers electrons from formate via the menaquinone pool to NapA independently of a membrane-bound c-type cytochrome of the NapC family. The napB and napD gene products are essential for nitrate respiration. NapD is required for the production of mature NapA. NapF or NapL function in NapA assembly and/or export
-
-
?
nitrate + reduced acceptor
nitrite + oxidized acceptor
show the reaction diagram
nitrate + reduced benzyl viologen
nitrite + benzyl viologen
show the reaction diagram
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen
show the reaction diagram
-
-
-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
show the reaction diagram
nitrate + reduced methyl viologen
nitrite + methyl viologen
show the reaction diagram
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen
show the reaction diagram
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
show the reaction diagram
-
the purified enzyme does not react with duroquinol or NADH. It may be that the purified enzyme has lost some of the components that mediate the electron transport from the physiological reductant to the catalytic 63000 Da polypeptide during the purification process
-
-
?
nitrate + reduced methylviologen
nitrite + oxidized methyl viologen + H2O
show the reaction diagram
nitrite + acceptor
nitrate + reduced acceptor
show the reaction diagram
reduced methyl viologen + chlorate
methyl viologen + chlorite
show the reaction diagram
reduced methyl viologen + nitrate
methyl viologen + nitrite
show the reaction diagram
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
nitrate + reduced acceptor
nitrite + acceptor
show the reaction diagram
nitrate + reduced acceptor
nitrite + acceptor + H2O
show the reaction diagram
-
Wolinella succinogenes transfers electrons from formate via the menaquinone pool to NapA independently of a membrane-bound c-type cytochrome of the NapC family. The napB and napD gene products are essential for nitrate respiration. NapD is required for the production of mature NapA. NapF or NapL function in NapA assembly and/or export
-
-
?
nitrate + reduced acceptor
nitrite + oxidized acceptor
show the reaction diagram
-
a model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex
-
-
?
nitrite + acceptor
nitrate + reduced acceptor
show the reaction diagram
additional information
?
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Bactopterin
-
common cofactor of eubacterial molybdoenzymes
cytochrome b
-
FAD
-
assimilatory nitrate reductases
heme b
heme c
iron-sulfur centre
molybdenum bis-molybdopterin guanine dinucleotide
-
one bis-MGD cofactor in a single polypeptide chain of 723 amino acids, extends across the interior of the molecule interacting with residues from all 4 domains, catalytic molybdenum site is coordinated to two MGD cofactors, Cys140 and a water/hydroxo ligand
molybdenum cofactor
molybdo-(bismolybdopterin)guanine dinucleotide
-
Mo-bis-MGD, NapA of the periplasmic nitrate reductase, assimilatory nitrate reductases
molybdo-bis(pyranopterin guanine dinucleotide)
-
molybdopterin guanine dinucleotide
NAD(P)H
[4Fe-4S]-center
-
additional information
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
iron-sulfur centre
KCl
-
maximum activity occurrs in the absence of NaCl and decreases with increasing concentrations of NaCl so that the activity is 46% of maximum when the enzyme is assayed in the presence of 4 M NaCl. Replacing NaCl with KCl does not significantly change the response
Molybdenum
Tungsten
-
0.06 mol tungsten per mol of enzyme complex
WO42-
-
enzyme purified from cells grown with 4.5 µM WO42- contains W as the metal cofactor. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor
[3Fe-4S] center
-
the amount of iron determined was consistent with the presence of one [3Fe-4S] center and four [4Fe-4S] centers
[4Fe-4S] center
-
the amount of iron determined was consistent with the presence of one [3Fe-4S] center and four [4Fe-4S] centers
additional information
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-n-heptyl-4-hydroxyquinoline N-oxide
-
only effective as inhibitor with the three-subunit enzyme and duroquinol as substrate
ammonium sulfate
-
ammonium sulfate and other ammonium salts inhibit
bathophenanthroline
-
uncompetitive inhibition
bathophenanthroline-sulfonate
-
uncompetitive inhibition
bromate
-
competitive inhibitor, 1 mM, 50% inhibition
Chlorate
cyanide
Dithiol
-
0.1 mM, 95% inhibition
Dithionite
-
1 mM, 24% loss of activity
dithiothreitol
EDTA
-
1 mM, 22% loss of activity
ferricyanide
-
at pH 10.5 the as prepared enzyme is inactivated
IO3-
-
1 mM, 40% inhibition
KClO4
-
1 mM, 33% inhibition
Mepacrine
-
1 mM, 40% inhibition
metronidazole
-
2 mM metronidazole inhibits nitrate reduction by 6.7% in the whole-cell lysate
NaCN
-
0.04 mM, complete inhibition
NaN3
-
0.04 mM, 59% inhibition
NH4HCO3
-
3.87 mM, complete inhibition
Nitrofurantoin
-
0.3 mM nitrofurantoin inhibits nitrate reduction by 19.5% in the whole-cell lysate
o-phenanthroline
-
3 mM, 25% inhibition
oxygen
p-chloromercuribenzoate
Sodium azide
-
-
Sodium cyanide
-
-
Sulfide
Thiocyanate
additional information
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
deoxycholate
-
activates
ferricyanide
-
enzyme as prepared is slowly activated at pH 7.2 over 250% of the original activity, dependent on ferricyanide concentration, little activation at pH 9.5
isopentenyladenine
nitrate reductase expression and activity in both roots and shoots of pineapple are significantly enhanced by isopentenyladenine
isopentenyladenine riboside
nitrate reductase expression and activity in both roots and shoots of pineapple are significantly enhanced by isopentenyladenine riboside
K3Fe(CN)6
-
the enzyme, inactive in vivo, may be reactivated in vitro by oxidation with K3Fe(CN)6
KNO3
-
10 mM, up to 4fold activation
nitrate
nitrite
-
the activity of the 125000 Da complex increases strikingly from 0.1 mM to 1 mM nitrite added, however, within the higher range of available nitrite, the intensity of this form decreases dramatically, whereas the activity of the 140000 Da band starts to grow in strength
p-chloromercuribenzoate
-
enhances enzyme activity at 0.1 mM and lower concentrations
zeatin
nitrate reductase expression and activity in both roots and shoots of pineapple are significantly enhanced by zeatin
zeatin riboside
nitrate reductase expression and activity in both roots and shoots of pineapple are significantly enhanced by zeatin riboside
additional information
-
4fold activation by heating to 75°C for 75 min
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0022 - 0.0029
benzyl viologen
0.138 - 5
Chlorate
0.25
methyl viologen
-
-
0.0032 - 5
nitrate
0.0002 - 1.6
reduced benzyl viologen
0.0028 - 1.5
reduced methyl viologen
additional information
additional information
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1350
Chlorate
Pyrobaculum aerophilum
-
at 75°C
2.5 - 1162
nitrate
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00013 - 0.55
azide
0.5 - 0.8
bathophenanthroline
1.3 - 2.4
bathophenanthroline-sulfonate
0.8 - 1.7
Chlorate
0.044 - 0.5
cyanide
0.16 - 0.28
Sulfide
1.4
Thiocyanate
-
nitrate as substrate
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0182 - 0.036
azide
0.0134 - 0.11
cyanide
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0003
-
nitrate, 10 mM NH4+, NADPH, aerobic
0.0057
-
nitrate, medium without nitrogen, NADPH, aerobic
0.0129
-
nitrate, 10 mM NO3-, NADPH, aerobic
0.0186
-
nitrate, 10 mM NH4+, methyl viologen, aerobic; nitrate, 10 mM NH4+, NADPH, anaerobic
0.02 - 0.04
-
rate of physiological reduction, lower than that obtained with reduced benzyl viologen
0.02
-
nitrate, medium without nitrogen, methyl viologen, aerobic
0.066
-
nitrate, medium without nitrogen, NADPH, anaerobic
0.0705
-
nitrate, 10 mM NO3-, methyl viologen, aerobic; nitrate, 10 mM NO3-, NADPH, anaerobic
0.0754
-
nitrate, 10 mM NH4+, methyl viologen, anaerobic
0.2659
-
nitrate, medium without nitrogen, methyl viologen, anaerobic
0.342
-
methyl viologen as electron donor, aerobic growth conditions, assay in intact cells
0.39
-
nitrate/nitrite reductase complex
0.423
-
nitrate, 10 mM NO3-, methyl viologen, anaerobic
0.523
-
benzyl viologen as electron donor, aerobic growth conditions, assay in intact cells
1.407
-
benzyl viologen as electron donor, anaerobic growth conditions, assay in intact cells
2.018
-
methyl viologen as electron donor, anaerobic growth conditions, assay in intact cells
2.157
-
methyl viologen as electron donor, microaerobic growth conditions, assay in intact cells
2.264
-
benzyl viologen as electron donor, microaerobic growth conditions, assay in intact cells
2.63
-
enzyme in cell-free extract
24.5
-
nitrate reductase I
35
P85098 and P85097
after 76fold purification
39.1
-
nitrate reductase II
62
-
37°C, pH 7.2
160
-
at 30°C, both enzyme forms, reduced benzyl viologen as reductant
229
-
at 30°C
326
-
nitrate as substrate, at 75°C
378
-
chlorate as substrate, at 75°C
2530
-
pH 7.4, 30°C
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3
-
nitrate as substrate
6.4
-
chlorate as substrate
6.8
-
broad pH-optimum from pH 5.8 to pH 7.1 with maximum at pH 6.8
7.1
-
nitrate as substrate
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
-
the activities at pH 6.0 and pH 8.0 are 83% and 81% of the activity at pH 7.2
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
-
assay at
32
activity assay; activity assay; activity assay; activity assay; activity assay
56
-
presence of 0.5 mM MgCl2
70
-
at 3.4 M NaCl
73
-
presence of 2 M KCl
85
-
presence of 4.27 M NaCl
95
-
highest activity at or above
additional information
-
temperature optimum is a function of both: the concentration and the specific cation present, increasing NaCl and KCl concentrations result in an increase in the maximal activity at higher temperatures
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70 - 80
-
maximal activity
additional information
-
-
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1
-
calculated
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
NapG and NapH proteins form a membrane-bound protein complex that is likely to catalyse menaquinol oxidation and electron transport to the periplasmic NapAB nitrate reductase complex
-
Manually annotated by BRENDA team
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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)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Rhodobacter sphaeroides (strain ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16000
-
x * 62000 + x * 52000 + x * 36000 + x * 16000, SDS-PAGE
19000
-
x * 150000 alpha + x * 60000 beta + x * 19000 gamma, alpha-subunit with catalytic function containing molybdenum cofactor and non-heme iron, beta-subunit is possibly involved in membrane binding, gamma-subunit is found in some preparations and is a b-type cytochrome
21000
-
alpha,beta,gamma, 1 * 127000 + 1 * 61000 + 1 * 21000, SDS-PAGE
23000
-
enzyme form predominating in the membrane fraction, SDS-PAGE
25000
-
x * 103000, x * 53000, x * 25000, SDS-PAGE
26000
-
gel filtration
28690
calculated, NarJ
31000
-
1 * 100000 + 1 * 60000 + 1 * 31000
36000
-
x * 62000 + x * 52000 + x * 36000 + x * 16000, SDS-PAGE
42000
-
x * 150000 + x * 58000 + x * 42000, SDS-PAGE
44000
-
alpha,beta, 1 * 150000 + 1 * 44000, SDS-PAGE
46060
calculated, NarK
53000
-
x * 103000, x * 53000, x * 25000, SDS-PAGE
60840
calculated, NarH
61000
-
alpha,beta,gamma, 1 * 127000 + 1 * 61000 + 1 * 21000, SDS-PAGE
63000
-
4 * 63000
67000
-
x * 150000 alpha + x * 67000 beta1 + x * 65000 beta2, molar ratio alpha:beta1 + beta2 is 1:1, SDS-PAGE
82500
-
x * 83400, deduced from gene sequence, x * 82500, SDS-PAGE, recombinant enzyme
86000
x * 17000 + x * 86000, SDS-PAGE
91000
-
x * 91000 + x * 17000, SDS-PAGE
100000
-
1 * 100000 + 1 * 60000 + 1 * 31000
103000
-
x * 103000, x * 53000, x * 25000, SDS-PAGE
116000
-
2 * 116000 + 2 * 60000, SDS-pAGE
117000
118000
-
x * 118000 + x * 62000, SDS-PAGE
120000
-
alpha,beta, 1 * 120000 + 1 * 60000, alpha subunit: catalytic subunit, beta subunit: a membrane attachment protein, SDS-PAGE
125000
-
enzyme form with remarkably increased activity, SDS-PAGE
127000
-
alpha,beta,gamma, 1 * 127000 + 1 * 61000 + 1 * 21000, SDS-PAGE
130000
136000
-
1 * 136000 + 1 * 65000 + 1 * 20000, SDS-PAGE
137000
P85098 and P85097
purified homogenous alpha subunit, non-denaturing PAGE
140000
-
active enzyme form, SDS-PAGE
141400
calculated, NarG
142000
-
4 * 142000 + 4 * 58000, subunits are probably associated in form of a double tetrahedron, SDS-PAGE
150000
165000
-
mean value, real value between 155000 Da and 175000 Da, PAGE
176000
180000 - 220000
P85098 and P85097
purified native enzyme, gel filtration
180000
190000
-
in the nitrate-fed cells, the 190000 Da form is the most abundant, SDS-PAGE
196000
-
always present in a monomeric form, PAGE
200000
208000
-
gel filtration
214000
-
three-subunit complex, calculated as the sum of the MWs of the subunits
220000
-
monomer form of the enzyme, analytical ultracentrifugation in presence of 0.2% deoxycholate
230000
P85098 and P85097
non-denaturing PAGE
235000
-
sucrose density gradient sedimentation
240000
-
gel filtration
260000
-
nitrate reductase I, abc2, monomeric form, gel filtration
265000
-
nitrate reductase from aerobic variant, estimated by gel filtration on a Toyopearl HW-55 column
290000
320000
-
in absence of deoxycholate, associated form, gel filtration
355000
-
nitrate reductase from anaerobic variant, estimated by gel filtration on a Toyopearl HW-55 column
380000
-
gel filtration
400000
-
13.9 S dimeric state, gel filtration
620000
720000
-
gel filtration
773000
-
analytical ultracentrifugation
880000
-
associated, probably tetrameric, form of enzyme
1000000
-
-
1060000
-
nitrate reductase I, (abc2)4, tetrameric form, gel filtration
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
heterotetramer
-
-
heterotrimer
-
respiratory nitrate reductases composed of the subunits NarG, 112-140 kDa, NarH, 52-64 kDa and NarI, 19-25 kDa
hexadecamer
-
4 * 117000 + 4 * 57000 + 8 * 52000, nitrate reductase II, SDS-PAGE
monomer
multimer
octamer
tetramer
trimer
additional information
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
proteolytic modification
additional information
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion method. Structure of proteolyzed form of recombinant NapB at 1.25 A resolution
-
crystal structure determined at a resolution of 3.2 A
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 10
-
stable
395168
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
-
immobilized enzyme more stable at 6°C than at 23°C
25
-
stable for mor than 30 h
65
-
10 min, stable
80
-
0.17 M NaCl: 90% loss of activity after 1 min, 0.85 M NaCl: 50% loss of activity after 7 min, 4.27-5.31 M NaCl: no loss of activity after 15 min, high salt concentrations protect enzyme against heat inactivation at 80°C due to a tighter, more stable configuration
100
-
half-life of 1.5 h, half-life within cell-membranes is 6 h, lipid environment stabilizes
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
deoxycholate stabilizes
-
freezing rapidly inactivates enzyme at a concentration of 0.1 mg per ml, 0.1 mM dithiothreitol stabilizes
-
immobilized enzyme more labile than free enzyme
-
NaCl protects against heat inactivation, 4.27-5.31 M NaCl
-
nitrate reductase II, which lacks the 52 kDa subunit, is much more labile than nitrate reductase I, deoxycholate stabilizes nitrate reductase II
-
slow freezing inactivates, but rapid freezing in liquid N2 and thawing at room temperature can be repeated 10 times without effect on enzyme activity, when reduced benzyl viologen is used as reductant
-
sucrose stabilizes
-
the enzyme is most stable in the absence of salt
-
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-19°C, 3 months, enzyme loses no activity
-
-70°C, in glycerol, stable for at least 3 months
-
20°C, 0.07 M imidazole buffer, pH 8.1, 24 h, nitrate reductase I: 45% loss of activity, probably due to loss of 52 kDa subunit and conversion to nitrate reductase II, nitrate reductase II: 95% loss of activity
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4°C, 0.07 M imidazole buffer, pH 8.1, 24 h, nitrate reductase I: 10% loss of activity, nitrate reductase II: 35% loss of activity
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4°C, 3-4 weeks, remains active
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4°C, 8 days, in the absence of NaCl, little if any loss of activity
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4°C, aerobic or anaerobic storage, absence of deoxycholate: per 24 h, 30% inactivation, presence of 0.2% deoxycholate: per 24 h, 10% inactivation
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4°C, phosphate buffer, pH 7.2 or Tris-HCl buffer, pH 8.0, enzyme concentration less than 0.5 mg/ml: half live is 5 days under strict anaerobic conditions and 3 days in air, enzyme concentration above 10 mg/ml: under argon, 20 days, no loss of activity
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4°C, prolonged storage, stable, when assayed with reduced benzyl viologen as reductant
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5°C, 0.1 M Tris-HCl buffer, pH 8.8, 10% sucrose, 24 h, 10% loss of activity, without sucrose: 75% loss of activity
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liquid N2 temperature, 6 months, stable
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room-temperature, many weeks, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
100fold purification
-
112fold purification
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120fold purification
-
134fold purification
-
137fold purification
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140fold purification
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2 enzyme forms with different subunit compositions
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2 forms: nitrate reductase I, nitrate reductase II
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2 forms: nitrate reductase I: 36.6fold purification, nitrate reductase II: 58.4fold purification
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40fold purification
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53fold purification, alphabeta enzyme, cytochrome b1 is present up to gel filtration in Sephacryl 200 during purification
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56fold purification
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85fold purification
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ammonium sulfate precipitation
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ammonium sulfate precipitation, Macro-Prep High Q column chromatography, HiTrap Phenyl FF column chromatography, Sephadex G-25 gel filtration, and Sephacryl S-300 gel filtration
P85098 and P85097
gamma-subunit of enzyme is lost during gel filtration purification, resulting in a cytochrome-free enzyme; partial
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His-tagged enzyme
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partial purification
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partial purification of the nitrate/nitrite reductase complex
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purification of the catalytic alpha-subunit of nitrate reductase A
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strain X5119
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the cell extract is centrifuged and the supernatant is used as source for the nitrate reductase
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using non-denaturating preparative electrophoresis in 7.5% polyacrylamide gel with constant buffer elution
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the narG gene, encoding the large alpha-subunit gene of enzyme, nucleotide sequence of part of nar DNA and sequence of N-terminal 147 amino acids of the alpha-subunit
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construction of napKEFD-lacZ translational gene fusions, dissection of the upstream region of napK of a nap-lacZ fusion plasmid, construction of the napC::omega-Smr/Spr insertion strain
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construction of transcriptional fusion constructs carrying 5' truncations and generation of individual single-point mutations in the full-length promotor sequence, generating full-length promoter fragments harbouring lesions
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napABC genes, encoding periplasmic nitrate reductase, are isolated and sequenced, NapB has 160 amino acides, NapC with 206 amino acids and a hydrophobic membrane-spanning domain near its N-terminus
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NarC, a 27 kDa membrane cytochrome c, is encoded as the first gene of the narCGHJIK1K2 operon for nitrate respiration and plays an essential role in the synthesis of active enzyme and for the attachment of enzyme to the membrane, sequence of narC, NarG is unable to bind to the cytoplasmic membrane in absence of NarC
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the membrane-bound nitrate reductase genes, nar genes, are cloned into the p-SCRIPT and pGEM-T vector for sequencing; the membrane-bound nitrate reductase genes, nar genes, are cloned into the p-SCRIPT and pGEM-T vector for sequencing; the membrane-bound nitrate reductase genes, nar genes, are cloned into the p-SCRIPT and pGEM-T vector for sequencing; the membrane-bound nitrate reductase genes, nar genes, are cloned into the p-SCRIPT and pGEM-T vector for sequencing; the membrane-bound nitrate reductase genes, nar genes, are cloned into the p-SCRIPT and pGEM-T vector for sequencing
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C181S
-
complete loss of nitrate reductase activity; inactive mutant enzyme
D196G
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complete loss of nitrate reductase activity; inactive mutant enzyme
D196G/E197A
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complete loss of nitrate reductase activity
E197A
-
mutant enzyme with reduced activity; reduced nitrate reductase activity
M182H
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complete loss of nitrate reductase activity; inactive mutant enzyme
R421E
-
complete loss of nitrate reductase activity
R421ED196G/E197A
-
inactive mutant enzyme
R421K
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mutant enzyme with reduced activity; reduced nitrate reductase activity
H49C
loss of catalytic activity. The midpoint potential value of the [4Fe-4] cluster is decreased by at least 500 mV
H49S
loss of catalytic activity. Both the [4Fe-4S] cluster and the molybdo-bis(pyranopterin guanine dinucleotide) cofactor are absent
R94S
residue in the vicinity of the [4Fe-4S] cluster. The midpoint potential value of the [4Fe-4] cluster is decreased by 115 mV, with a concomitant decrease in enzyme turnover to 30% of the wild type
H49C
-
loss of catalytic activity. The midpoint potential value of the [4Fe-4] cluster is decreased by at least 500 mV
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H49S
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loss of catalytic activity. Both the [4Fe-4S] cluster and the molybdo-bis(pyranopterin guanine dinucleotide) cofactor are absent
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R94S
-
residue in the vicinity of the [4Fe-4S] cluster. The midpoint potential value of the [4Fe-4] cluster is decreased by 115 mV, with a concomitant decrease in enzyme turnover to 30% of the wild type
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additional information
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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inoculated of root nodules of Glycine max with Bradyrhizobium japonicum. In the presence of nitrate, all of the nitrosylleg-hemoglobin within normoxic nodules arises from nitrate reduction by the bacterial enzyme, whereas the enzyme is only responsible for half of the nitrosylleg-hemoglobin within hypoxic nodules
medicine
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potential target for persistence-specific anti-tubercular drug development
synthesis
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strong correlation of enzyme transcription rate and activity, enzyme activity is highest when growth occurs on butyrate followed by acetate and succinate
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Show AA Sequence (7416 entries)
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LINKS TO OTHER DATABASES (specific for EC-Number 1.7.99.4)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
SYSTERS
Protein Mutant Database
InterPro (database of protein families, domains and functional sites)