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Literature summary 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.
    View publication on PubMed

Cloned(Commentary)

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

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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

EC Number Inhibitors Comment Organism Structure
1.13.11.50 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
1.13.11.50 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
1.13.11.50 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
1.13.11.50 additional information no inactivation effect of Fe3+ Acinetobacter johnsonii
1.13.11.50 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
1.13.11.50 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

EC Number Metals/Ions Comment Organism Structure
1.13.11.50 Cu2+ sulfate, similar binding as Fe2+ in wild-type enzyme Acinetobacter johnsonii
1.13.11.50 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
1.13.11.50 Fe3+ citrate, no interference with Fe2+ Acinetobacter johnsonii
1.13.11.50 Mn2+ sulfate, similar binding as Fe2+ in wild-type enzyme Acinetobacter johnsonii
1.13.11.50 Ni2+ sulfate, similar binding as Fe2+ in wild-type enzyme Acinetobacter johnsonii
1.13.11.50 Zn2+ sulfate, similar binding as Fe2+ in wild-type enzyme Acinetobacter johnsonii

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.13.11.50 18000
-
 SDS-PAGE Acinetobacter johnsonii

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.50 Acinetobacter johnsonii Q8GNT2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.50 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.13.11.50 0.09
-
 wild-type enzyme, 20 mM Tris/HCl buffer, pH 7.5, 25°C Acinetobacter johnsonii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.50 3,4-dihydroxyphenylacetate + O2
-
 Acinetobacter johnsonii ?
-
 ?
1.13.11.50 pentane-2,4-dione + O2
-
 Acinetobacter johnsonii methylglyoxal + acetate
-
 ?
1.13.11.50 potassium oxalate + O2
-
 Acinetobacter johnsonii ?
-
 ?
1.13.11.50 quercetin + O2
-
 Acinetobacter johnsonii ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.50 homotetramer
-
 Acinetobacter johnsonii

Synonyms

EC Number Synonyms Comment Organism
1.13.11.50 cupin-type dioxygenase
-
 Acinetobacter johnsonii
1.13.11.50 Dke1
-
 Acinetobacter johnsonii

Cofactor

EC Number Cofactor Comment Organism Structure
1.13.11.50 heme necessary for enzyme activity Acinetobacter johnsonii