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Literature summary extracted from

  • Lin, Y.; St Maurice, M.
    The structure of allophanate hydrolase from Granulibacter bethesdensis provides insights into substrate specificity in the amidase signature family (2013), Biochemistry, 52, 690-700.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.54 gene CGDNIH1, expression of N-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain HMS174 (DE3) Granulibacter bethesdensis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.54 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

EC Number Protein Variants Comment Organism
3.5.1.54 R307A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 R307M site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299A/R307A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299A/R307M site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299F/R307A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis
3.5.1.54 Y299F/R307M site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Granulibacter bethesdensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.54 additional information
-
 additional information kinetics of wild-type and mutant enzymes, overview Granulibacter bethesdensis
3.5.1.54 0.1
-
 allophanate pH 7.4, 25°C, recombinant wild-type enzyme Granulibacter bethesdensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.1.54 65000
-
 x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE Granulibacter bethesdensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.54 allophanate + H2O Granulibacter bethesdensis
-
 2 CO2 + 2 NH3
-
 ?
3.5.1.54 allophanate + H2O Granulibacter bethesdensis ATCC BAA-1260
-
 2 CO2 + 2 NH3
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.54 Granulibacter bethesdensis Q0BRB0 gene CGDNIH1
-
3.5.1.54 Granulibacter bethesdensis ATCC BAA-1260 Q0BRB0 gene CGDNIH1
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.54 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.54 allophanate + H2O
-
 Granulibacter bethesdensis 2 CO2 + 2 NH3
-
 ?
3.5.1.54 allophanate + H2O the enzyme exhibits high specificity for allophanate Granulibacter bethesdensis 2 CO2 + 2 NH3
-
 ?
3.5.1.54 allophanate + H2O
-
 Granulibacter bethesdensis ATCC BAA-1260 2 CO2 + 2 NH3
-
 ?
3.5.1.54 allophanate + H2O the enzyme exhibits high specificity for allophanate Granulibacter bethesdensis ATCC BAA-1260 2 CO2 + 2 NH3
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.54 ? x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE Granulibacter bethesdensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.54 25
-
 assay at Granulibacter bethesdensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.54 18.2
-
 allophanate pH 7.4, 25°C, recombinant wild-type enzyme Granulibacter bethesdensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.54 7.4
-
 assay at Granulibacter bethesdensis

General Information

EC Number General Information Comment Organism
3.5.1.54 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
3.5.1.54 metabolism allophanate hydrolase catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2 Granulibacter bethesdensis
3.5.1.54 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 [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.54 0.0000017
-
 allophanate pH 7.4, 25°C, mutant R307A Granulibacter bethesdensis
3.5.1.54 0.0000036
-
 allophanate pH 7.4, 25°C, mutant R307M Granulibacter bethesdensis
3.5.1.54 0.0000071
-
 allophanate pH 7.4, 25°C, mutant Y299A/R307M Granulibacter bethesdensis
3.5.1.54 0.000019
-
 allophanate pH 7.4, 25°C, mutant Y299F/R307M Granulibacter bethesdensis
3.5.1.54 0.000027
-
 allophanate pH 7.4, 25°C, mutant Y299A/R307A Granulibacter bethesdensis
3.5.1.54 0.0024
-
 allophanate pH 7.4, 25°C, mutant Y299A Granulibacter bethesdensis
3.5.1.54 0.0035
-
 allophanate pH 7.4, 25°C, mutant Y299F Granulibacter bethesdensis
3.5.1.54 182
-
 allophanate pH 7.4, 25°C, recombinant wild-type enzyme Granulibacter bethesdensis