Cloned (Comment) | Organism |
---|---|
expression of Strep-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Acinetobacter johnsonii |
Protein Variants | Comment | Organism |
---|---|---|
F115A | site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme | Acinetobacter johnsonii |
F119A | site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme | Acinetobacter johnsonii |
F59A | site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme | Acinetobacter johnsonii |
Y70F | site-directed mutagenesis, the mutant shows reduced turnover and altered iron binding compared to the wild-type enzyme | Acinetobacter johnsonii |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state and single-turnover kinetics of substrate binding and conversion in wild-type Dke1 and mutants, overview | Acinetobacter johnsonii |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | nonheme Fe(II) cofactor, distinct organization of the hydrophilic triad in the free and substrate-ligated wild-type enzyme. In the free species, the Fe(II) center is coordinated to three histidines and one glutamate, whereas the substrate-ligated, catalytically competent enzyme-substrate complex has an Fe(II) center with three-histidine coordination, with a small fraction of three-histidine, one-glutamate coordination | Acinetobacter johnsonii | |
additional information | spectrophotometrical monitoring, Fe2+ and Fe3+ coordination, formation of iron(II) enzyme-substrate complexes, and detachment kinetics, overview | Acinetobacter johnsonii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
pentane-2,4-dione + O2 | Acinetobacter johnsonii | - |
acetate + methylglyoxal | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acinetobacter johnsonii | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography | Acinetobacter johnsonii |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
1,1-difluoropentane-2,4-dione + O2 | - |
Acinetobacter johnsonii | ? | - |
? | |
pentane-2,4-dione + O2 | - |
Acinetobacter johnsonii | acetate + methylglyoxal | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | each subunit is organized in a single-domain beta-barrel fold | Acinetobacter johnsonii |
Synonyms | Comment | Organism |
---|---|---|
diketone cleaving enzyme | - |
Acinetobacter johnsonii |
Dke1 | - |
Acinetobacter johnsonii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Acinetobacter johnsonii |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0015 | - |
1,1-difluoropentane-2,4-dione | mutant Y70F, pH 7.5, 25°C | Acinetobacter johnsonii | |
0.008 | - |
1,1-difluoropentane-2,4-dione | mutant F59A, pH 7.5, 25°C | Acinetobacter johnsonii | |
0.018 | - |
1,1-difluoropentane-2,4-dione | mutant F115A, pH 7.5, 25°C | Acinetobacter johnsonii | |
0.022 | - |
1,1-difluoropentane-2,4-dione | mutant F119A, pH 7.5, 25°C | Acinetobacter johnsonii | |
0.036 | - |
1,1-difluoropentane-2,4-dione | wild-type enzyme, pH 7.5, 25°C | Acinetobacter johnsonii | |
0.7 | - |
Pentane-2,4-dione | mutant F59A, pH 7.5, 25°C | Acinetobacter johnsonii | |
1.27 | - |
Pentane-2,4-dione | mutant Y70F, pH 7.5, 25°C | Acinetobacter johnsonii | |
1.4 | - |
Pentane-2,4-dione | mutant F115A, pH 7.5, 25°C | Acinetobacter johnsonii | |
3.2 | - |
Pentane-2,4-dione | mutant F119A, pH 7.5, 25°C | Acinetobacter johnsonii | |
6.6 | - |
Pentane-2,4-dione | wild-type enzyme, pH 7.5, 25°C | Acinetobacter johnsonii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Acinetobacter johnsonii |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the cupin superfamily of proteins | Acinetobacter johnsonii |
additional information | Dke1 displays an atypical three-histidine metal binding site. Role of the protein structure in the catalysis of beta-diketone cleavage at the threehistidine metal center of diketone cleaving enzyme by computational methods in correlation with kinetic and mutational analyses. Molecular dynamics simulations, using quantum mechanically deduced parameters for the nonheme Fe(II) cofactor. Distinct organization of the hydrophilic triad in the free and substrate-ligated wild-type enzyme. In the free species, the Fe(II) center is coordinated to three histidines and one glutamate, whereas the substrate-ligated, catalytically competent enzyme-substrate complex has an Fe(II) center with three-histidine coordination, with a small fraction of three-histidine, one-glutamate coordination | Acinetobacter johnsonii |