Information on EC 1.13.11.50 - acetylacetone-cleaving enzyme

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The expected taxonomic range for this enzyme is: Acinetobacter johnsonii

EC NUMBER
COMMENTARY hide
1.13.11.50
-
RECOMMENDED NAME
GeneOntology No.
acetylacetone-cleaving enzyme
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
pentane-2,4-dione + O2 = acetate + methylglyoxal
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C-C-bond cleavage
-
-
oxidation
redox reaction
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
acetylacetone:oxygen oxidoreductase
An Fe(II)-dependent enzyme. Forms the first step in the acetylacetone degradation pathway of Acinetobacter johnsonii. While acetylacetone is by far the best substrate, heptane-3,5-dione, octane-2,4-dione, 2-acetylcyclohexanone and ethyl acetoacetate can also act as substrates.
CAS REGISTRY NUMBER
COMMENTARY hide
524047-53-8
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
the enzyme belongs to the cupin superfamily of proteins
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
1,1,1-trifluoro-2,4-pentanedione + O2
?
show the reaction diagram
-
-
-
-
?
1,1,1-trifluoro-pentane-2,4-dione + O2
?
show the reaction diagram
1,1-difluoro-2,4-pentanedione + O2
?
show the reaction diagram
-
-
-
-
?
1,1-difluoropentane-2,4-dione + O2
?
show the reaction diagram
-
-
-
-
?
1-phenyl-1,3-butanedione + O2
?
show the reaction diagram
-
-
-
-
?
1-phenylbutane-1,3-dione + O2
?
show the reaction diagram
-
-
-
-
?
2,4-dioxopentanoic acid ethyl ester + O2
?
show the reaction diagram
2,4-nonadione + O2
?
show the reaction diagram
-
poor substrate
-
-
?
2,4-octanedione + O2
?
show the reaction diagram
-
-
-
-
?
2-acetylcyclohexanone + O2
?
show the reaction diagram
-
-
-
-
?
3,4-dihydroxyphenylacetate + O2
?
show the reaction diagram
-
-
-
?
3,5-heptanedione + O2
?
show the reaction diagram
-
-
-
-
?
3-methyl-2,4-pentanedione + O2
?
show the reaction diagram
-
-
-
-
?
3-oxobutanone + O2
?
show the reaction diagram
4,4-difluoro-1-phenyl-1,3-butanedione + O2
?
show the reaction diagram
-
-
-
-
?
4-hydroxy-4-methyl-2-pentanone + O2
?
show the reaction diagram
-
-
-
-
?
5,5-dimethylhexane-2,4-dione + O2
?
show the reaction diagram
-
poor substrate
-
-
?
acetylacetone + O2
acetate + 2-oxopentanal
show the reaction diagram
-
-
-
-
?
acetylcyclohexanone + O2
?
show the reaction diagram
-
poor substrate
-
-
?
ethylacetoacetate + O2
?
show the reaction diagram
-
-
-
-
?
pentane-2,4-dione + O2
acetate + 2-oxopropanal
show the reaction diagram
pentane-2,4-dione + O2
acetate + methylglyoxal
show the reaction diagram
-
-
-
-
?
pentane-2,4-dione + O2
methylglyoxal + acetate
show the reaction diagram
-
-
-
?
potassium oxalate + O2
?
show the reaction diagram
-
-
-
?
quercetin + O2
?
show the reaction diagram
-
-
-
?
additional information
?
-
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
acetylacetone + O2
acetate + 2-oxopentanal
show the reaction diagram
-
-
-
-
?
pentane-2,4-dione + O2
acetate + 2-oxopropanal
show the reaction diagram
pentane-2,4-dione + O2
acetate + methylglyoxal
show the reaction diagram
-
-
-
-
?
pentane-2,4-dione + O2
methylglyoxal + acetate
show the reaction diagram
Q8GNT2
-
-
-
?
additional information
?
-
-
acetylacetone dioxygenase catalyzes the dioxygen-dependent degradation of beta-dicarbonyl compounds
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
heme
-
necessary for enzyme activity
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
sulfate, similar binding as Fe2+ in wild-type enzyme
Fe3+
-
citrate, no interference with Fe2+
Iron
-
dependent on, metalloenzyme, 1 iron bound per subunit, required for positioning of the substrate and for rendering of the appropriate electronic environment
Mn2+
-
sulfate, similar binding as Fe2+ in wild-type enzyme
Ni2+
-
sulfate, similar binding as Fe2+ in wild-type enzyme
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
-
20 mM Tris/HCl buffer, pH 7.5, 25C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
EDTA
-
largely irreversible losses
hexacyanoferrate(III)
-
2.5 mM, 80% decrease in activity within 10 min
KCN
-
largely irreversible losses
Mn2+
-
20 mM Tris/HCl buffer, pH 7.5, 25C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Ni2+
-
20 mM Tris/HCl buffer, pH 7.5, 25C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
o-phenanthroline
-
largely irreversible losses
Zn2+
-
20 mM Tris/HCl buffer, pH 7.5, 25C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enzyme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
additional information
-
no inactivation effect of Fe3+
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0025
1,1,1-trifluoro-2,4-pentanedione
-
apparent value at 25C
0.0027
1,1-difluoro-2,4-pentanedione
-
apparent value at 25C
0.002
4,4-difluoro-1-phenyl-1,3-butanedione
-
apparent value at 25C
0.0091
acetylacetate
-
-
0.26
O2
-
pH 7.5, 25C
0.000009 - 0.0091
Pentane-2,4-dione
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0049
1,1,1-trifluoro-2,4-pentanedione
Acinetobacter johnsonii
-
apparent value at 25C
0.0043 - 6.6
1,1,1-trifluoro-pentane-2,4-dione
0.036
1,1-difluoro-2,4-pentanedione
Acinetobacter johnsonii
-
apparent value at 25C
0.0015 - 0.036
1,1-difluoropentane-2,4-dione
0.00043
4,4-difluoro-1-phenyl-1,3-butanedione
Acinetobacter johnsonii
-
apparent value at 25C
8.5
acetylacetate
Acinetobacter johnsonii
-
-
0.7 - 8.5
Pentane-2,4-dione
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.09
-
wild-type enzyme, 20 mM Tris/HCl buffer, pH 7.5, 25C
1.34
-
recombinant enzyme, 35C
additional information
-
different assay methods
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65000
-
gel filtration
66400
-
SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
half-life 308 min in a membrane bioreactor, increase in stability from 5C to 10C
35
-
half-life 9 min in a membrane bioreactor
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
soluble enzyme is poorly stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity column chromatography
-
purified, concentrated enzyme is dialyzed twice against 2 mM EDTA in 20 mM Tris/HCl (pH 7.5) to strip off iron, incubation in 2 mM metal ion as sulfate salt plus ascorbate to prevent Fe2+ oxidation, unbound metal ions are removed by 3 cycles of gel filtration using nucleic acid purification column
-
recombinant Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography
-
Sepharose Q fast flow column chromatography Superdex 200 gel filtration, Resource Q column chromatography, Nap-5 column gel filtration, and Phenyl Sepharose HP chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli strain BL21(DE3)
-
expression in Escherichia coli strain BL21(DE3)
-
expression of mutant gene vector in Escherichia coli BL21(DE3)
-
expression of Strep-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E69Q
-
lower thermal stability of beta-sheet secondary structure, half catalytic center activity and remarkably silent difference in apparent substrate binding compared to the wild type enzyme
E98A
-
site-direted mutagenesis
E98Q
-
site-direted mutagenesis
F115A
-
site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme
F119A
-
site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme
F59A
-
site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme
H104E
-
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
H104N
-
approximately 1% of the specific activity of wild-type enzyme with substrate pentan-2,4-dione, retains binding affinity for Fe2+ at binding site I, binding seems to be tighter than in wild-type, small effect on other metal ions
H62E
-
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
H62N
-
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, binding disruption of Cu2+, Mn2+, and Ni2+ compared with wild-type
H64D
-
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
H64E
-
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
H64N
-
conversion of substrate in a strictly Fe2+-concentration dependent manner, substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
R80A
-
site-direted mutagenesis
R80A/E98A
-
site-direted mutagenesis
T107A
-
site-direted mutagenesis
Y70A
-
site-direted mutagenesis
Y70A/R80A/E98A
-
site-direted mutagenesis
Y70F/R80A/E98A
-
site-direted mutagenesis
additional information
-
the exchange of 3 histidines in the Fe2+-binding centre shows that these histidines are crucial for for binding Fe2+ and for Fe2+-dependent dioxygenase activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
environmental protection
-
biodegradation by the enzyme of the widely used industrial chemical acetylacetone, i.e. 2,4-pentanedione, which has toxic effects, in a membrane bioreactor, determination of operational stability of the enzyme in the reactor at different temperatures, simulations