Cloned (Comment) | Organism |
---|---|
expression of mutant gene vector in Escherichia coli BL21(DE3) | Acinetobacter johnsonii |
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 | 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 | 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 Maximum [Da] | Comment | Organism |
---|---|---|---|
18000 | - |
SDS-PAGE | Acinetobacter johnsonii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
pentane-2,4-dione + O2 | Acinetobacter johnsonii | - |
methylglyoxal + acetate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acinetobacter johnsonii | Q8GNT2 | - |
- |
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 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 | 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 | Comment | Organism |
---|---|---|
homotetramer | - |
Acinetobacter johnsonii |
Synonyms | Comment | Organism |
---|---|---|
cupin-type dioxygenase | - |
Acinetobacter johnsonii |
Dke1 | - |
Acinetobacter johnsonii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
heme | necessary for enzyme activity | Acinetobacter johnsonii |