Literature summary for 3.5.1.77 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.

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
P178C	half-life at 50°C is nearly identical to wild-type enzyme	Agrobacterium tumefaciens
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]

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

Organism

Organism	UniProt	Comment	Textmining
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	-	?

Synonyms

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

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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
63	-	63.1°C, 20 min, 50% loss of activity, wild-type enzyme	Agrobacterium tumefaciens
63	-	63.5°C, 20 min, 50% loss of activity, mutant enzyme A222C	Agrobacterium tumefaciens
64	-	63.6°C, 20 min, 50% loss of activity, mutant enzyme P178C	Agrobacterium tumefaciens
65	-	64.8°C, 20 min, 50% loss of activity, mutant enzyme P295C/F304C	Agrobacterium tumefaciens
68	-	Tm-value of mutant enzyme P178C: 68.3°C, Tm-value of mutant enzyme A222C: 67.8°C	Agrobacterium tumefaciens
69	-	Tm-value of wild-type enzyme: 68.6°C	Agrobacterium tumefaciens
71	-	20 min, 50% loss of activity, mutant enzyme A302C	Agrobacterium tumefaciens
72	-	Tm-value of mutant enzyme P295C/F304C: 72.3°C	Agrobacterium tumefaciens
77	-	Tm-value of mutant enzyme A302C: 77.4°C	Agrobacterium tumefaciens

Turnover Number [1/s]

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