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Literature summary for 3.5.1.77 extracted from

  • Oh, K.H.; Nam, S.H.; Kim, H.S.
    Improvement of oxidative and thermostability of N-carbamyl-D-amino acid amidohydrolase by directed evolution (2002), Protein Eng., 15, 689-695.
    View publication on PubMed

Protein Variants

Protein Variants Comment Organism
G75S 96% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 42.5% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens
H58Y 80% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 20.6% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens
M184L 80% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 20% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens
M184L/T262A 46% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 54.8% compared to 5% for the wild-type enzyme. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 58.6% of the wild-type ratio Agrobacterium tumefaciens
Q23L 81% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 63.9% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens
Q23L/V40A/H58Y/G75S/M184L/T262A mutant enzyme with improved oxidative and thermostability, 79.3% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 72.4% of the wild-type ratio Agrobacterium tumefaciens
T262A 49% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 40.9% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens
V40A 93% loss of activity of mutant enzyme M184L after 30 min at 70°C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25°C is 20.6% compared to 5% for the wild-type enzyme Agrobacterium tumefaciens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C, wild-type enzyme Agrobacterium tumefaciens
1.1
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C,mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A Agrobacterium tumefaciens
2.4
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C, mutant enzyme M184L, T262A Agrobacterium tumefaciens

Organism

Organism UniProt Comment Textmining
Agrobacterium tumefaciens
-
NRRL B11291
-
Agrobacterium tumefaciens NRRL B11291
-
NRRL B11291
-

Oxidation Stability

Oxidation Stability Organism
residual activity after incubation with 0.2 mM hydrogen peroxide for 30 min at 25°C is 5% for the wild-type enzyme, 20% for mutant enzyme M184L, 40.9% for mutant enzyme T262A, 54.8% for mutant enzyme M184L/T262A, 63.9% for mutant enzyme Q23L, 20.6% for mutant enzymes V40A and H58Y, 42.5% for mutant enzyme G75S, 79.3% for mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A Agrobacterium tumefaciens

Purification (Commentary)

Purification (Comment) Organism
-
Agrobacterium tumefaciens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-carbamoyl-D-p-hydroxyphenylglycine + H2O
-
Agrobacterium tumefaciens D-p-hydroxyphenylglycine + NH3 + CO2
-
?
N-carbamoyl-D-p-hydroxyphenylglycine + H2O
-
Agrobacterium tumefaciens NRRL B11291 D-p-hydroxyphenylglycine + NH3 + CO2
-
?

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
70
-
30 min, 90% loss of wild-type activity, 80% loss of activity of mutant enzyme M184L, 49% loss of activity of mutant enzyme T262A, 46% loss of activity of mutant enzyme M184L/T262A 81% loss of activity of mutant enzyme Q23L, 93% loss of activity of mutant enzyme V40A, 80% loss of activity of mutant enzyme H58Y, 96% loss of activity of mutant enzyme G75S, 21% loss of activity of mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A Agrobacterium tumefaciens
73
-
30 min, mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A, 50% loss of activity Agrobacterium tumefaciens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.9
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C, mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A Agrobacterium tumefaciens
9.5
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C, wild-type enzyme Agrobacterium tumefaciens
12.3
-
N-carbamoyl-D-p-hydroxyphenylglycine pH 7.0, 40°C, mutant enzyme M184L, T262A Agrobacterium tumefaciens