Information on EC 1.19.6.1 - nitrogenase (flavodoxin)

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

EC NUMBER
COMMENTARY hide
1.19.6.1
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RECOMMENDED NAME
GeneOntology No.
nitrogenase (flavodoxin)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4 reduced flavodoxin + N2 + 16 ATP + 16 H2O = 4 oxidized flavodoxin + H2 + 2 NH3 + 16 ADP + 16 phosphate
show the reaction diagram
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
Metabolic pathways
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Microbial metabolism in diverse environments
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nitrogen fixation II (flavodoxin)
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Nitrogen metabolism
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nitrate assimilation
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SYSTEMATIC NAME
IUBMB Comments
reduced flavodoxin:dinitrogen oxidoreductase (ATP-hydrolysing)
Requires Mg2+. It is composed of two components, dinitrogen reductase and dinitrogenase, that can be separated but are both required for nitrogenase activity. Dinitrogen reductase is a [4Fe-4S] protein, which, at the expense of ATP, transfers electrons from a dedicated flavodoxin to dinitrogenase. Dinitrogenase is a protein complex that contains either a molybdenum-iron cofactor, a vanadium-iron cofactor, or an iron-iron cofactor, that reduces dinitrogen in three succesive two-electron reductions from nitrogen to diimine to hydrazine to two molecules of ammonia. The reduction is initiated by formation of hydrogen. The enzyme can also reduce acetylene to ethylene (but only very slowly to ethane), azide to nitrogen and ammonia, and cyanide to methane and ammonia. In the absence of a suitable substrate, hydrogen is slowly formed. Some enzymes utilize ferredoxin rather than flavodoxin as the electron donor (see EC 1.18.6.1, nitrogenase).
CAS REGISTRY NUMBER
COMMENTARY hide
9013-04-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain DJ995
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Manually annotated by BRENDA team
Japanese trefoil
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Manually annotated by BRENDA team
cv. Aragon R-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
6 reduced flavodoxin + N2 + 6 H2O + 6 ATP
6 oxidized flavodoxin + 2 NH3 + 6 H+ + 6 ADP + 6 phosphate
show the reaction diagram
dithionite + H+ + N2 + ATP
?
show the reaction diagram
N2H2 + H+
NH3
show the reaction diagram
reduced ferredoxin + H+ + N2 + ATP
oxidized ferredoxin + H2 + NH3 + ADP + phosphate
show the reaction diagram
reduced flavodoxin + H+ + acetylene + ATP
oxidized flavodoxin + ethylene + ADP + phosphate
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
6 reduced flavodoxin + N2 + 6 H2O + 6 ATP
6 oxidized flavodoxin + 2 NH3 + 6 H+ + 6 ADP + 6 phosphate
show the reaction diagram
reduced ferredoxin + H+ + N2 + ATP
oxidized ferredoxin + H2 + NH3 + ADP + phosphate
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FeMo cofactor
FeMo protein
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a complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, Fe6 within FeMo-cofactor provides the unique site for alkyne substrate binding and has van der Waals contact with the side chains of alpha-Val70l and alpha-Gln191, overview
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flavodoxin
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MoFe protein
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two parallel electron channels may exist between the [8Fe-7S] cluster and FeMo-cofactor
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Molybdenum
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
diazene
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inhibits proton reduction
H2
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inhibits both N2 and diazene reduction
NIFI1,2
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a regulatory protein, inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein, NifI1,2 inhibits ATP- and MoFe protein-dependent oxidation of the Fe protein, and NIFI1,2 binding prevents association of the two nitrogenase components, the inhibition is relieved by 2-oxoglutarate. NIFI1,2 is unable to bind to an AlF4- stabilized Fe protein:MoFe protein complex. Both nifI1 and nifI2 are required for regulation in vivo
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nitrate
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2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide suppresses the inhibition by nitrate
O2
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complete reversible inhibition, reversibility decreases by increasing the time of exposure to O2
sodium nitroprusside
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inhibits acetylene-reduction activity of nitrogenase by 1 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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steady-state turnover analysis with diazene and hydrazine bound to the FeMo cofactor, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
400
flavodoxin hydroquinone
Azotobacter vinelandii
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before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.027
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reductant dithionite
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at, acetylene reduction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
230000
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nitrogenase MoFe protein component
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * alpha-beta-monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 2.17 A resolution. The structure displays typical structural motifs shared by other nitrogen-fixaion flavodoxins, a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50’s loop and a remarkable difference in the electrostatic potential surface with respect to non-nitrogenfixation flavodoxins
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
complete reversible inhibition by O2, reversibility decreases by increasing the time of exposure to O2, after 20 min 60% reversibility of the inhibition remains
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440140
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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gene nifD, expression of wild-type and mutant enzymes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
alphaH195Q
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site-directed mutagenesis, the mutant shows an altered electron transfer substrate specificity compared to the wild-type enzyme,overview
alphaQ191K
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site-directed mutagenesis, the mutant shows reduced electron transfer activity compared to the wild-type enzyme, it reduces only H+, not N2, overview
Q191A/V70A
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site-directed mutagenesis, the double mutation does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene
V70A
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site-directed mutagenesis, substitution of alpha-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne
V70G
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site-directed mutagenesis, the mutant MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne, but no detectable reduction of the internal alkyne 2-butyne
V70I
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site-directed mutagenesis, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene
Q191A/V70A
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site-directed mutagenesis, the double mutation does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene
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V70A
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site-directed mutagenesis, substitution of alpha-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne
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V70G
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site-directed mutagenesis, the mutant MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne, but no detectable reduction of the internal alkyne 2-butyne
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V70I
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site-directed mutagenesis, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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nitrogenase activity decline due to salinity stress is significantly lower and delayed in plants nodulated by flavodoxin-expressing bacteria than after nodulation by wild-type bacteria. After 3 days of stress, this decrease is approximately 60% for nodules elicited by wild-type bacteria and only 40% for flavodoxin-expressing nodules. Overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions