Information on EC 1.7.7.2 - ferredoxin-nitrate reductase

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

EC NUMBER
COMMENTARY hide
1.7.7.2
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RECOMMENDED NAME
GeneOntology No.
ferredoxin-nitrate reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrite + H2O + 2 oxidized ferredoxin = nitrate + 2 reduced ferredoxin + 2 H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrate reduction VI (assimilatory)
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nitrate assimilation
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Nitrogen metabolism
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
nitrite:ferredoxin oxidoreductase
A molybdenum-iron-sulfur protein.
CAS REGISTRY NUMBER
COMMENTARY hide
60382-69-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 7119
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Manually annotated by BRENDA team
strain ATCC 29413
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Manually annotated by BRENDA team
strains 7101 and 7601
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Manually annotated by BRENDA team
heterocystous nitrogen-fixing cyanobacterial strains
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Manually annotated by BRENDA team
strain 7119
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Manually annotated by BRENDA team
strain N1, phototrophic, purple sulfur bacterium
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Manually annotated by BRENDA team
strain N1, phototrophic, purple sulfur bacterium
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Manually annotated by BRENDA team
strain 1829
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Manually annotated by BRENDA team
strain 1829
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Manually annotated by BRENDA team
I3R9M9: alpha-subunit (narG), I3R9M8: beta subunit (narH)
I3R9M9 and I3R9M8
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain CU 1462/7
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Manually annotated by BRENDA team
strain CU 1462/7
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Manually annotated by BRENDA team
formerly known as Anacystis nidulans
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Manually annotated by BRENDA team
strain WH 8102
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Manually annotated by BRENDA team
strain 6803, unicellular cyanobacterium
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Manually annotated by BRENDA team
Thermosynechococcus sp.
strain BP-1
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Manually annotated by BRENDA team
strain BP-1
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Manually annotated by BRENDA team
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
chlorite + acceptor
show the reaction diagram
chlorate + reduced methylviologen
chlorite + H2O + oxidized methylviologen
show the reaction diagram
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6% of kcat with nitrate
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?
nitrate + 2 reduced ferredoxin + 2 H+
nitrite + H2O + 2 oxidized ferredoxin
show the reaction diagram
nitrate + reduced ferredoxin
nitrite + H2O + oxidized ferredoxin
show the reaction diagram
nitrate + reduced ferredoxin
nitrite + oxidized ferredoxin
show the reaction diagram
nitrate + reduced flavodoxin
nitrite + H2O + oxidized flavodoxin
show the reaction diagram
nitrate + reduced flavodoxin
nitrite + oxidized flavodoxin + H2O
show the reaction diagram
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flavodoxin can physiologically substitute for ferredoxin under conditions of iron starvation
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nitrate + reduced methyl viologen
nitrite + H2O + oxidized methyl viologen
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 methylviologen
nitrite + H2O + oxidized methylviologen
show the reaction diagram
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?
nitrite + acceptor
nitrate + reduced acceptor
show the reaction diagram
selenate + reduced methylviologen
selenite + H2O + oxidized methylviologen
show the reaction diagram
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1% of kcat with nitrate
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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
nitrate + 2 reduced ferredoxin + 2 H+
nitrite + H2O + 2 oxidized ferredoxin
show the reaction diagram
nitrate + reduced ferredoxin
nitrite + H2O + oxidized ferredoxin
show the reaction diagram
nitrate + reduced ferredoxin
nitrite + oxidized ferredoxin
show the reaction diagram
nitrate + reduced flavodoxin
nitrite + oxidized flavodoxin + H2O
show the reaction diagram
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flavodoxin can physiologically substitute for ferredoxin under conditions of iron starvation
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additional information
?
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the regulation of the nar gene occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
heme c
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enzyme contains 1.5 heme molecules of the c-type
iron-sulfur centre
molybdenum cofactor
molybdopterin
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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stimulates activity about 30%
Fe2+
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stimulates activity about 50%
iron-sulfur centre
Mg2+
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stimulates activity about 30%
Mn2+
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stimulates activity about 50%, stimulates reaction rate and level of nitrite formation
Molybdenum
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonia
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after initial slight stimulation rapid and complete inhibition, promotes conversion of ferredoxin-nitrate reductase into its reduced inactive form, metabolic interconversion
azide
Chlorate
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competitive inhibitor with respect to nitrate
cyanide
Dithionite
FAD
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photoactivated FAD, irreversible inactivation
KCl
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inhibition of nitrate reductase-ferredoxin complex formation at high ionic strength
light
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light- and oxygen-dependent inactivation of enzyme activity in absence but not in presence of any added nitrogen source, inactivation may result from an oxidative modification of enzyme, assay in cell suspension
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MgSO4
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inhibition of nitrate reductase-ferredoxin complex formation at high ionic strength
N-acetylsuccinimide
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115fold excess, loss of more than 80% and 5-10% loss of ferredoxin-dependent and methylviologen dependent activity after 1 h, respectively
NaCl
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more than 80% inhibition above 200 mM, inhibition of nitrate reductase-ferredoxin complex formation at high ionic strength
oxygen
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light- and oxygen-dependent inactivation of enzyme activity in absence but not in presence of any added nitrogen source, inactivation may result from an oxidative modification of enzyme, assay in cell suspension
p-hydroxymercuribenzoate
Phenylglyoxal
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76fold excess, approx. 60% and 20% loss of ferredoxin dependent and methylviologen dependent activity after 2 h, respectively
sulfite
1 mM, 30% inhibition
Thiocyanate
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10 mM, 53% inhibition. Preincubating for 1 h does not increase the inhibition
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cyanate
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05
Ferredoxin
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spinach ferredoxin
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0.25
methyl viologen
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0.045 - 2.1
nitrate
0.013 - 0.038
reduced ferredoxin
0.0028 - 2.5
reduced methyl viologen
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
80 - 1090
nitrate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0007 - 13
azide
0.8
Chlorate
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37°C, pH 7.2
0.5
cyanide
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37°C, pH 7.2
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.2
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in situ assay, cells grown on media containing nitrate
62
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37°C, pH 7.2
305
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ferredoxin-linked activity
1020
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methyl viologen-linked activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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anaerobic assay, methyl viologen- and ferredoxin-linked activity
9.5 - 10.5
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reduced methyl viologen as electron donor
10.2
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aerobic assay, methyl viologen-linked activity
10.5
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dithionite-reduced methyl viologen as electron donor
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
pH 5.0: about 50% of maximal activity, pH 11.0: about 80% of maximal activity
6 - 8
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the activities at pH 6.0 and pH 8.0 are 83% and 81% of the activity at pH 7.2
7.3 - 9.2
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at pH 7.3 and 9.2: 50% of activity maximum, aerobic assay, methyl viologen-linked and ferredoxin-linked activity
8.3 - 9.8
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at pH 8.3 and 9.8: about 50% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
the enzyme shows a remarkable thermophilicity and works well up to 50°C in the presence of high salt concentrations
60
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for NaCl concentrations of 1.3 and 0.9 M
70
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at 3.4 M NaCl
80
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for NaCl concentrations of 3.1 and 2.2 M
additional information
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.52
calculated from amino acid sequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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enzyme functions in the soluble state in the cytoplasm
Manually annotated by BRENDA team
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recombinant enzyme activity is found exclusively in the soluble fraction
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Manually annotated by BRENDA team
additional information
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activity remains associated with photosynthetic subcellular particles
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
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1 * 105000 + 1 * 50000, SDS-PAGE
60000
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2 * 116000 + 2 * 60000, SDS-pAGE
78000
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x * 78000, SDS-PAGE
83000
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1 * 83000, SDS-PAGE
90000
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ultracentrifugation
100000
105000
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1 * 105000 + 1 * 50000, SDS-PAGE
116000
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2 * 116000 + 2 * 60000, SDS-pAGE
132000
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gel filtration
380000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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5 min, crude extract, stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
low ionic strength, 70 h, 0.002 mg protein/ml, 10 mM phosphate buffer or 50 mM Tris-HCl buffer, pH 7.5, unstable
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NaCl stabilizes, after 43 h, with 2.7 M, 2.1 M or 0.9 M NaCl: 95% loss of activity, after 90 h, with 4.3 M NaCl: completely stable
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the enzyme is most stable in the absence of salt
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
reversible inactivation of ferredoxin-linked activity by exposure of dithionite solution of the enzyme to air, but no effect on methyl viologen-linked activity
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440443
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-19°C, 3 months, enzyme loses no activity
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-20°C, 20% glycerol, several months, no loss of activity
-20°C, in 50 mM Tris-HCl buffer, pH 7.5, 0.5 M NaCl, 1 mM EDTA, 1 month, no loss of activity
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4°C, 50 mM phosphate buffer, pH 7, 20% glycerol, 2 weeks, no loss of activity
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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, activity decreases about 40% in 1 week when the crude extract is stored. At temperatures higher than 4°C, the activity depletion in the crude extract is 60–80%
4°C, several weeks, no loss of activity
4°C, the activity of the pure sample is stable for 2–3 weeks
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
177fold purification
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185fold partial purification
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19000fold purification
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partial purification
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purified from a few cyanobacterial strains
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recombinant enzyme
recombinant His-tagged nitrate reductase, Ni2+-affinity column
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recombinant nitrate reductase
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression of His-tagged nitrate reductase in Escherichia coli
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expression of native and His-tagged nitrate reductase in Escherichia coli
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narB structural gene for nitrate reductase is cloned and expressed in Escherichia coli as a 76 kDa polypeptide with 729 amino acids
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strain 7120: gene encoding nitrate reductase is cloned and its complete amino acid sequence deduced
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
activity is detected between 72 and 168 h of incubation, and it reaches the maximum value when the absorbance of the culture is around 0.47 (at 600 nm). This maximum activity value is observed shortly after the beginning of the exponential phase of growth, and in that moment, high nitrite concentration within the media is quantified (up to 18.8 mM). This growth phase is characterised by oxygen depletion (culture medium is initially aerobic, but it becomes microaerobic as soon as the biomass increases shortly before the stationary phase of growth)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C12A
no actiivty, no iron-sulfur cluster
C16S
1.9% of wild type activity with ferredoxin, no iron-sulfur cluster
C56A
no activity, no iron-sulfur cluster
C56S
70% of wild type activity with ferredoxin, contains an iron-sulfur cluster
C9A
no activity, no iron-sulfur cluster
C9H
no narB expression