Information on EC 1.14.14.10 - nitrilotriacetate monooxygenase

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

EC NUMBER
COMMENTARY hide
1.14.14.10
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RECOMMENDED NAME
GeneOntology No.
nitrilotriacetate monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrilotriacetate + FMNH2 + H+ + O2 = iminodiacetate + glyoxylate + FMN + H2O
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrilotriacetate degradation
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SYSTEMATIC NAME
IUBMB Comments
nitrilotriacetate,FMNH2:oxygen oxidoreductase (glyoxylate-forming)
Requires Mg2+. The enzyme from Aminobacter aminovorans (previously Chelatobacter heintzii) is part of a two component system that also includes EC 1.5.1.42 (FMN reductase), which provides reduced flavin mononucleotide for this enzyme.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Chelativorans sp.
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Manually annotated by BRENDA team
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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
EDTA + O2 + FMNH2 + H+
ethylenediaminetriacetate + glyoxylate + H2O + FMN
show the reaction diagram
Chelativorans sp.
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?
nitrilotriacetate + FMNH2 + H+ + O2
iminodiacetate + glyoxylate + FMN + H2O
show the reaction diagram
nitrilotriacetate + O2 + FMNH2 + H+
iminodiacetate + glyoxylate + H2O + FMN
show the reaction diagram
Chelativorans sp.
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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
nitrilotriacetate + FMNH2 + H+ + O2
iminodiacetate + glyoxylate + FMN + H2O
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FMN
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omission of FMN or replacement of FMN by FAD results in a residual activity of 2% when component B is not resaturated with FMN
FMNH2
NADH
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NADPH cannot replace NADH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Al3+
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nitrilotriacetate is only a substrate when complexed with cations such as Mg2+, Al3+, Cu2+, Ni2+, Zn2+, Fe2+ or Co2+
Cu2+
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nitrilotriacetate is only a substrate when complexed with cations such as Mg2+, Al3+, Cu2+, Ni2+, Zn2+, Fe2+ or Co2+
Fe2+
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nitrilotriacetate is only a substrate when complexed with cations such as Mg2+, Al3+, Cu2+, Ni2+, Zn2+, Fe2+ or Co2+
Mn2+
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Mn2+ ions are able to replace Mg2+ but lead to a higher uncoupled NADH oxidation and enzyme activity is reduced to 70% of that with MgCl2
Ni2+
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nitrilotriacetate is only a substrate when complexed with cations such as Mg2+, Al3+, Cu2+, Ni2+, Zn2+, Fe2+ or Co2+
Zn2+
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nitrilotriacetate is only a substrate when complexed with cations such as Mg2+, Al3+, Cu2+, Ni2+, Zn2+, Fe2+ or Co2+
additional information
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no nitrilotriacetate consumption is observed with Ca2+,Fe2+, Fe3+, Zn2+, Cu2+, or Ni2+; the enzyme contains less than 0.15 atom of Fe per mol of protein, indicating that neither Fe-sulfur clusters nor cytochromes are present
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoate)
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67% residual activity at 1 mM
Ca2+
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5% residual activity at 10 mM
EDTA
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3% residual activity at 10 mM
FAD
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24% residual activity at 0.1 mM
NaCl
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54% residual activity at 250 mM
Ni2+
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ionic or uncomplexed Ni2+ (2 mM) is inhibitory to the enzyme
p-hydroxymercuribenzoate
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12% residual activity at 1 mM
Zn2+
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ionic or uncomplexed Zn2+ (2 mM) is inhibitory to the enzyme
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
component A of NTA monooxygenase
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the ability of component B of NTA monooxygenase to catalyze the oxidation of nitrilotriacetate to iminodiacetate and glyoxylate is completely dependent on the presence of component A
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FMN
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NTA-Mo activity increases by a factor of 4 when 0.003 mM FMN is included in the assay mixture, there is 0.4 mol of FMN per mol of purified component B
additional information
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the activity is increased neither by additions of 20% (w/v) polyethylene glycol or sorbitol
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0013
FMN
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in 40 mM TAPS buffer at pH 8.5 and 25°C
0.0317 - 0.5
nitrilotriacetate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.076
in 30 mM Tris-HCl (pH 8.0), 10 mM FMN, and 2 mM MgCl2, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
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pH optimum for NTA consumption in cell extracts. At pH 7.2 and 8.4, 25% of the maximum activity is obtained, and no activity is left at pH 6.0 or 9.0
8.5
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pH optimum for the purified enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.6
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35% activity of the purified enzyme remains at pH 7.0 and 9.6
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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12% activity is left at 2°C, and no activity is observed at 40°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
calculated from amino acid sequence
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34500
x * 34500, calculated from amino acid sequence
36000
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2 * 36000, component B of NTA monooxygenase, SDS-PAGE
45000
Chelativorans sp.
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gel filtration
47800
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2 * 47800, estimated from SDS-PAGE
50500
x * 50500, calculated from amino acid sequence
100000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
Chelativorans sp.
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1 * 45000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 1.6 M NH2SO4, 0.1 M NaCl and 0.1 M HEPES pH 7.5
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sitting drop vapor diffusion method, using 0.2 M magnesium chloride, 0.1 M MES pH 6.0 and 20% PEG 6000
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme activity is rather unstable in crude cell extract, even in the presence of the protease inhibitors leupeptin and pepstatin
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, crude cell extract, up to 1 month, no loss of activity
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-70°C, purified component B of NTA monooxygenase in 20 mM HEPES buffer with 2 mM dithiothreitol, at pH 7.8, several months, without any significant loss of activity
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-80°C, partially purified, dialyzed enzyme, 3 months, no loss of activity
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4°C, crude cell extract, 48 hours, 50% loss of activity
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4°C, crude extract, 50 mM Tris-HCl, 200 h, 50% loss of activity
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4°C, purified component B of NTA monooxygenase in 20 mM HEPES buffer at pH 7.8, 7 days, 70% loss of activity
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4°C, purified component B of NTA monooxygenase in 20 mM HEPES buffer with 2 mM dithiothreitol and ammonium sulfate to 5% saturation, at pH 7.8, 7 days, 5% loss of activity
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4°C, purified component B of NTA monooxygenase in 20 mM HEPES buffer with 2 mM dithiothreitol, at pH 7.8, 7 days, 20% loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, Blue 3GA agarose column chromatography, MonoQ HR 5/5 column chromatography, and hydroxyapatite column chromatography
Chelativorans sp.
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ammonium sulfate precipitation, phenyl Sepharose column chromatography, TMAE-Fractogel column chromatography, and phenyl Superose column chromatography
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HisTrap FF column chromatography and Superdex 75 gel filtration
Ni-NTA column chromatography, HiTrap Q anion-exchange column chromatography
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partially purified by ammonium sulfate precipitation and phenyl agarose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) Rosetta cells
expressed in Escherichia coli DH5alpha cells
expressed in Escherichia coli JM105 cells
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induction of nitrilotriacetate-degrading enzyme proceeds faster when the culture is supplied with mixtures of D-glucose and nitrilotriacetate rather than with nitrilotriacetate alone
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induction of nitrilotriacetate-degrading enzyme proceeds faster when the culture is supplied with mixtures of D-glucose and nitrilotriacetate rather than with nitrilotriacetate alone; synthesis of nitrilotriacetate monooxygenase becomes induced when nitrilotriacetate contributes to more than approximately 1-3% of the total carbon in the substrate mixture supplied
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starvation of nitrilotriacetate-grown cells leads to a loss of nitrilotriacetate monooxygenase protein. A transition from nitrilotriacetate to D-glucose is accompanied by a rapid loss of nitrilotriacetate monooxygenase
synthesis of nitrilotriacetate monooxygenase becomes induced when nitrilotriacetate contributes to more than approximately 1-3% of the total carbon in the substrate mixture supplied
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