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Literature summary extracted from

  • Chiu, W.C.; You, J.Y.; Liu, J.S.; Hsu, S.K.; Hsu, W.H.; Shih, C.H.; Hwang, J.K.; Wang, W.C.
    Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase (2006), J. Mol. Biol., 359, 741-753.
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.77 hanging drop vapour diffusion method using protein solutions containing 15 mg/ml protein with precipitating solution at room temperature. A222C and A302C mutants are initially grown as micro-crystals with the precipitating condition of 1.2 M lithium sulfate and 0.1 M Hepes buffer (pH 7.0). A micro-seeding method is then applied to obtain large single crystals. For P95C/F304C and P178C, no crystals are obtained. Crystals structures of D-NCase mutants, A302C and A222C Agrobacterium tumefaciens

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.77 A222C crystal structure is nearly identical to wild-type enzyme, half-life at 50°C is 1.1fold higher than that of wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 A302C crystal structure is nearly identical to wild-type enzyme, 123.9% of wild-type activity, temperature-optimum is 10°C higher than that of wild-type enzyme, half-life at 50°C is 2.5fold higher than that of wild-type enzyme, 4.2fold increase in kcat/Km at 65°C, 1.5fold increase at 55°C and 1.15fold increase at 37°C Agrobacterium tumefaciens
3.5.1.77 P178C half-life at 50°C is nearly identical to wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 P295C/F304C 107.3% of wild-type activity, temperature-optimum is 5°C higher than that of wild-type enzyme, half-life at 50°C is 2fold higher than that of wild-type enzyme. 2.5fold increase in kcat/Km at 65°C, 1.1fold increase at 55°C and at 37°C Agrobacterium tumefaciens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.77 1.08
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 1.12
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 1.14
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 1.14
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 1.16
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 1.22
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 1.24
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 1.32
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 1.47
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.77 Agrobacterium tumefaciens
-
-
-

Substrates and Products (Substrate)

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

Synonyms

EC Number Synonyms Comment Organism
3.5.1.77 D-NCAase
-
 Agrobacterium tumefaciens
3.5.1.77 N-carbamoyl D-amino acid amidohydrolase
-
 Agrobacterium tumefaciens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.77 55
-
 wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 60
-
 mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 65
-
 mutant enzyme A302C Agrobacterium tumefaciens

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.5.1.77 50
-
 half-life: 8.1 h (wild-type enzyme), 20.4 h (mutant enzyme P295C/F304C), 16.2 h (mutant enzyme P295C/F304C), 8 h (mutant enzyme P178C), 9.0 h (mutant enzyme A222C) Agrobacterium tumefaciens
3.5.1.77 63
-
 63.1°C, 20 min, 50% loss of activity, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 63
-
 63.5°C, 20 min, 50% loss of activity, mutant enzyme A222C Agrobacterium tumefaciens
3.5.1.77 64
-
 63.6°C, 20 min, 50% loss of activity, mutant enzyme P178C Agrobacterium tumefaciens
3.5.1.77 65
-
 64.8°C, 20 min, 50% loss of activity, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 68
-
 Tm-value of mutant enzyme P178C: 68.3°C, Tm-value of mutant enzyme A222C: 67.8°C Agrobacterium tumefaciens
3.5.1.77 69
-
 Tm-value of wild-type enzyme: 68.6°C Agrobacterium tumefaciens
3.5.1.77 71
-
 20 min, 50% loss of activity, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 72
-
 Tm-value of mutant enzyme P295C/F304C: 72.3°C Agrobacterium tumefaciens
3.5.1.77 77
-
 Tm-value of mutant enzyme A302C: 77.4°C Agrobacterium tumefaciens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.77 0.03 0.55 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 0.031 0.51 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 0.52
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 1.1
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 2 8 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 6.93
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 7.73
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 7.88
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 7.97
-
 N-carbamoyl-D-p-hydroxyphenylglycine 37°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 13.71
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 14.88
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, wild-type enzyme Agrobacterium tumefaciens
3.5.1.77 15.25
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, mutant enzyme P295C/F304C Agrobacterium tumefaciens
3.5.1.77 19.08
-
 N-carbamoyl-D-p-hydroxyphenylglycine 55°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens
3.5.1.77 22.47
-
 N-carbamoyl-D-p-hydroxyphenylglycine 65°C, pH 7.0, mutant enzyme A302C Agrobacterium tumefaciens