Cloned (Comment) | Organism |
---|---|
gene oxdC, expression of the C-terminally His6-tagged protein in Escherichia coli strain BL21(DE3) | Bacillus subtilis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant mutants R92A, DELTA162-163, S161A, and E162A, hanging-drop vapour diffusion method at 18°C, the mutants enzymes generally crystallize with 8-15% v/v PEG 8000, 0.1 M Tris, pH 8.5, and 0-15% xylitol, further method optimization for each mutant enzyme, X-ray diffraction structure determination and analysis at 2.0-3.1 A resolution, molecular modelling | Bacillus subtilis |
Protein Variants | Comment | Organism |
---|---|---|
D297A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
E162A | site-directed mutagenesis, the mutant lacks both oxalate decarboxylase and oxalate oxidase activities, structure analysis | Bacillus subtilis |
E162D | site-directed mutagenesis, E162D mutant retains 29% of the decarboxylase activity compared to the wild-type enzyme | Bacillus subtilis |
E162Q | site-directed mutagenesis, E162Q mutant retains only 1% of the decarboxylase activity compared to the wild-type enzyme | Bacillus subtilis |
E333A | site-directed mutagenesis, the mutant shows reduced oxalate decarboxylase and oxalate oxidase activities compared to the wild-type enzyme | Bacillus subtilis |
E333Q | site-directed mutagenesis, the mutant lacks both oxalate decarboxylase and oxalate oxidase activities | Bacillus subtilis |
E33D | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
H299A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
additional information | construction of deletion mutant DELTA162-163 or inactive deletion mutant DELTA162-164, structure analysis, manganese content of mutant enzymes, overview | Bacillus subtilis |
R270A | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
R270Q | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
R92A | site-directed mutagenesis, the mutant lacks both oxalate decarboxylase and oxalate oxidase activities, structure analysis | Bacillus subtilis |
R92K | site-directed mutagenesis, the mutant lacks both oxalate decarboxylase and oxalate oxidase activities | Bacillus subtilis |
S161A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme, structure analysis | Bacillus subtilis |
S164A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Bacillus subtilis |
T165P | site-directed mutagenesis, altered comformation with dominant conformation of the lid compared to the wild-type enzyme | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetic analysis, different assay methods | Bacillus subtilis | |
1.14 | - |
oxalate | pH 4.0, 26°C, recombinant mutant R270Q | Bacillus subtilis | |
1.9 | - |
oxalate | pH 4.0, 26°C, recombinant mutant R92K | Bacillus subtilis | |
2.3 | - |
oxalate | pH 4.0, 26°C, recombinant deletion mutant DELTA163-163 | Bacillus subtilis | |
4.1 | - |
oxalate | pH 4.0, 26°C, recombinant mutant E333A | Bacillus subtilis | |
6.3 | - |
oxalate | pH 4.0, 26°C, recombinant mutant E333D | Bacillus subtilis | |
6.6 | 16.4 | oxalate | pH 4.0, 26°C, recombinant wild-type enzyme | Bacillus subtilis | |
8 | - |
oxalate | pH 4.0, 26°C, recombinant mutant R270A | Bacillus subtilis | |
11.2 | - |
oxalate | pH 4.0, 26°C, recombinant mutant D297A | Bacillus subtilis | |
11.6 | - |
oxalate | pH 4.0, 26°C, recombinant mutant H299A | Bacillus subtilis | |
13.5 | - |
oxalate | pH 4.0, 26°C, recombinant mutant E162Q | Bacillus subtilis | |
17.4 | - |
oxalate | pH 4.0, 26°C, recombinant mutant E162D | Bacillus subtilis | |
25 | - |
oxalate | pH 4.0, 26°C, recombinant mutant T165P | Bacillus subtilis | |
31 | - |
oxalate | pH 4.0, 26°C, recombinant mutant S164A | Bacillus subtilis | |
71 | - |
oxalate | pH 4.0, 26°C, recombinant mutant E161A | Bacillus subtilis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | required, each subunit contains two similar, but distinct, manganese sites 1 and 2, only site 1 is catalytically active, and site 2 is purely structural, manganese content of mutant enzymes, overview | Bacillus subtilis | |
O2 | required for catalysis | Bacillus subtilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
oxalate + H+ | Bacillus subtilis | - |
formate + CO2 | - |
ir | |
oxalate + H+ | Bacillus subtilis 168 | - |
formate + CO2 | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | O34714 | gene oxdC | - |
Bacillus subtilis 168 | O34714 | gene oxdC | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Bacillus subtilis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
activity of recombinant wild-type and mutant enzymes, overview | Bacillus subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the enzyme also shows oxalate oxidase activity, catalytic cycle, overview | Bacillus subtilis | ? | - |
? | |
additional information | the enzyme also shows oxalate oxidase activity, catalytic cycle, overview | Bacillus subtilis 168 | ? | - |
? | |
oxalate + H+ | - |
Bacillus subtilis | formate + CO2 | - |
ir | |
oxalate + H+ | catalytic cycle involving radical formation with O2, overview, only Mn2+ binding site 1 is catalytically active, while Mn2+ binding site 2 is purely structural, overview | Bacillus subtilis | formate + CO2 | - |
ir | |
oxalate + H+ | - |
Bacillus subtilis 168 | formate + CO2 | - |
ir | |
oxalate + H+ | catalytic cycle involving radical formation with O2, overview, only Mn2+ binding site 1 is catalytically active, while Mn2+ binding site 2 is purely structural, overview | Bacillus subtilis 168 | formate + CO2 | - |
ir |
Subunits | Comment | Organism |
---|---|---|
hexamer | homohexamer, each subunit contains two similar, but distinct, manganese sites, only site 1 is catalytically active and site 2 is purely structural | Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
26 | - |
assay at | Bacillus subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4 | - |
assay at | Bacillus subtilis |