Literature summary for 1.13.11.50 extracted from

Leitgeb, S.; Straganz, G.D.; Nidetzky, B.
Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii (2009), Biochem. J., 418, 403-411.

Search for more literature for 1.13.11.50

Cloned(Commentary)

Cloned (Comment)	Organism
expression of mutant gene vector in Escherichia coli BL21(DE3)	Acinetobacter johnsonii

Protein Variants

Protein Variants	Comment	Organism
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii
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	Acinetobacter johnsonii

Inhibitors

Inhibitors	Comment	Organism	Structure
Cu2+	20 mM Tris/HCl buffer, pH 7.5, 25°C, 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	Acinetobacter johnsonii
H2O2	1 M H2O2 causes complete loss of enzyme activity in less than 10 min, contains no Fe2+ (probably oxidized to Fe3+), partial reconstitution (40%) with 2 mM Fe2+, 20 mM Tris/HCl buffer, pH 7.5, 25°C	Acinetobacter johnsonii
Mn2+	20 mM Tris/HCl buffer, pH 7.5, 25°C, 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	Acinetobacter johnsonii
additional information	no inactivation effect of Fe3+	Acinetobacter johnsonii
Ni2+	20 mM Tris/HCl buffer, pH 7.5, 25°C, 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	Acinetobacter johnsonii
Zn2+	20 mM Tris/HCl buffer, pH 7.5, 25°C, 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	Acinetobacter johnsonii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Cu2+	sulfate, similar binding as Fe2+ in wild-type enzyme	Acinetobacter johnsonii
Fe2+	sulfate, necessary for enzyme activity. About 0.9 mol Fe2+/mol wild-type enzyme, less than 5% Fe2+ in mutants except 0.27 mol/mol H104E-enzyme and 0.45 mol/mol H104N-enzyme	Acinetobacter johnsonii
Fe3+	citrate, no interference with Fe2+	Acinetobacter johnsonii
Mn2+	sulfate, similar binding as Fe2+ in wild-type enzyme	Acinetobacter johnsonii
Ni2+	sulfate, similar binding as Fe2+ in wild-type enzyme	Acinetobacter johnsonii
Zn2+	sulfate, similar binding as Fe2+ in wild-type enzyme	Acinetobacter johnsonii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
18000	-	SDS-PAGE	Acinetobacter johnsonii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
pentane-2,4-dione + O2	Acinetobacter johnsonii	-	methylglyoxal + acetate	-	?

Organism

Organism	UniProt	Comment	Textmining
Acinetobacter johnsonii	Q8GNT2	-	-

Purification (Commentary)

Purification (Comment)	Organism
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	Acinetobacter johnsonii

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.09	-	wild-type enzyme, 20 mM Tris/HCl buffer, pH 7.5, 25°C	Acinetobacter johnsonii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
3,4-dihydroxyphenylacetate + O2	-	Acinetobacter johnsonii	?	-	?
pentane-2,4-dione + O2	-	Acinetobacter johnsonii	methylglyoxal + acetate	-	?
potassium oxalate + O2	-	Acinetobacter johnsonii	?	-	?
quercetin + O2	-	Acinetobacter johnsonii	?	-	?

Subunits

Subunits	Comment	Organism
homotetramer	-	Acinetobacter johnsonii

Synonyms

Synonyms	Comment	Organism
cupin-type dioxygenase	-	Acinetobacter johnsonii
Dke1	-	Acinetobacter johnsonii

Cofactor

Cofactor	Comment	Organism	Structure
heme	necessary for enzyme activity	Acinetobacter johnsonii

Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.

Release 2023.1 (January 2023)