Cloned (Comment) | Organism |
---|---|
gene CGDNIH1, expression of N-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain HMS174 (DE3) | Granulibacter bethesdensis |
Crystallization (Comment) | Organism |
---|---|
purified enzyme free or in complex with the substrate analog malonate, hanging-drop vapor diffusion method, mixing of 4 mg/ml protein in 10 mM HEPES, pH 8.0, 50 mM NaCl, and 1 mM DTT, with reservoir solution in a 1:1 ratio to a final volume of 0.005 ml, the latter containing 100 mM PIPES, pH 6.5, and 1.04 M sodium malonate, room temperature, 2 months, followed by microseeding, 5-15 days, X-ray diffraction structure determination and analysis at 2.2-2.8 A resolution, molecular replacement | Granulibacter bethesdensis |
Protein Variants | Comment | Organism |
---|---|---|
R307A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
R307M | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299A/R307A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299A/R307M | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299F | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299F/R307A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
Y299F/R307M | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Granulibacter bethesdensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics of wild-type and mutant enzymes, overview | Granulibacter bethesdensis | |
0.1 | - |
allophanate | pH 7.4, 25°C, recombinant wild-type enzyme | Granulibacter bethesdensis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
65000 | - |
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE | Granulibacter bethesdensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
allophanate + H2O | Granulibacter bethesdensis | - |
2 CO2 + 2 NH3 | - |
? | |
allophanate + H2O | Granulibacter bethesdensis ATCC BAA-1260 | - |
2 CO2 + 2 NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Granulibacter bethesdensis | Q0BRB0 | gene CGDNIH1 | - |
Granulibacter bethesdensis ATCC BAA-1260 | Q0BRB0 | gene CGDNIH1 | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His8-tagged wild-type and mutant enzymes from Escherichia coli strain HMS174 (DE3) by nickel affinity chromatography, dialysis, and anion exchange chromatography | Granulibacter bethesdensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
allophanate + H2O | - |
Granulibacter bethesdensis | 2 CO2 + 2 NH3 | - |
? | |
allophanate + H2O | the enzyme exhibits high specificity for allophanate | Granulibacter bethesdensis | 2 CO2 + 2 NH3 | - |
? | |
allophanate + H2O | - |
Granulibacter bethesdensis ATCC BAA-1260 | 2 CO2 + 2 NH3 | - |
? | |
allophanate + H2O | the enzyme exhibits high specificity for allophanate | Granulibacter bethesdensis ATCC BAA-1260 | 2 CO2 + 2 NH3 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE | Granulibacter bethesdensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Granulibacter bethesdensis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
18.2 | - |
allophanate | pH 7.4, 25°C, recombinant wild-type enzyme | Granulibacter bethesdensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Granulibacter bethesdensis |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cisSer-Lys catalytic triad | Granulibacter bethesdensis |
metabolism | allophanate hydrolase catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2 | Granulibacter bethesdensis |
additional information | Tyr299 and Arg307 seem to serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172, nucleophilic attack by serine results in a covalent tetrahedral intermediate that is stabilized by an oxyanion hole. After displacement of ammonia, the covalent intermediate is hydrolyzed to release the product. The unique C-terminal domain is conserved, but it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Active site architecture and structure-function analysis, overview | Granulibacter bethesdensis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0000017 | - |
allophanate | pH 7.4, 25°C, mutant R307A | Granulibacter bethesdensis | |
0.0000036 | - |
allophanate | pH 7.4, 25°C, mutant R307M | Granulibacter bethesdensis | |
0.0000071 | - |
allophanate | pH 7.4, 25°C, mutant Y299A/R307M | Granulibacter bethesdensis | |
0.000019 | - |
allophanate | pH 7.4, 25°C, mutant Y299F/R307M | Granulibacter bethesdensis | |
0.000027 | - |
allophanate | pH 7.4, 25°C, mutant Y299A/R307A | Granulibacter bethesdensis | |
0.0024 | - |
allophanate | pH 7.4, 25°C, mutant Y299A | Granulibacter bethesdensis | |
0.0035 | - |
allophanate | pH 7.4, 25°C, mutant Y299F | Granulibacter bethesdensis | |
182 | - |
allophanate | pH 7.4, 25°C, recombinant wild-type enzyme | Granulibacter bethesdensis |