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Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

Johansson, E.; Neuhard, J.; Willemoes, M.; Larsen, S.; Biochemistry 43, 6020-6029 (2004)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
3.5.4.5
overexpression of wild-type enzyme and mutant enzymes R56A, R56Q and C53H/R56Q in Escherichia coli
Bacillus subtilis
Crystallization (Commentary)
EC Number
Crystallization
Organism
3.5.4.5
hanging drop vapour diffusion method. R56A and R56Q crystallize in the same space group as the wild-type enzyme with two subunits in the asymmetric unit, whereas C53H/R56Q can not crystallize in this crystal form but is crystallized in another space group, P3(2)21, with a full tetramer in the asymmetric unit
Bacillus subtilis
Engineering
EC Number
Amino acid exchange
Commentary
Organism
3.5.4.5
C53H
attempts to express and purify the mutant enzyme are unsuccessful
Bacillus subtilis
3.5.4.5
C53H/R56Q
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Because of dissociation into its inactive subunits, it is impossible to determine the kinetic parameters for the mutant enzyme
Bacillus subtilis
3.5.4.5
R56A
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Similar KM-value but decreased Vmax-value compared to wild-type enzyme
Bacillus subtilis
3.5.4.5
R56D
zinc-binding capacity of mutant enzyme is reduced
Bacillus subtilis
3.5.4.5
R56Q
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Similar KM-value but decreased Vmax-value compared to wild-type enzyme
Bacillus subtilis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.4.5
0.0075
-
deoxycytidine
pH 7.8, 25°C, mutant enzyme R56A
Bacillus subtilis
3.5.4.5
0.029
-
deoxycytidine
pH 7.8, 25°C, mutant enzyme R56Q
Bacillus subtilis
3.5.4.5
0.14
-
cytidine
pH 7.6, 25°C, mutant enzyme R56A
Bacillus subtilis
3.5.4.5
0.216
-
cytidine
pH 7.6, 25°C, wild-type enzyme
Bacillus subtilis
3.5.4.5
0.236
-
deoxycytidine
pH 7.8, 25°C, wild-type enzyme
Bacillus subtilis
3.5.4.5
0.267
-
cytidine
pH 7.6, 25°C, mutant enzyme R56Q
Bacillus subtilis
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
3.5.4.5
Zinc
wild-type enzyme and mutant enzymes R56A, R56Q and C53H/R56Q contain about 1 mol of zinc per subunit. Mutant enzyme R56D contains 0.2 mol of zinc per subunit
Bacillus subtilis
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.5.4.5
Bacillus subtilis
P19079
-
-
Purification (Commentary)
EC Number
Commentary
Organism
3.5.4.5
-
Bacillus subtilis
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.4.5
cytidine + H2O
-
654757
Bacillus subtilis
uridine + NH3
-
-
-
?
3.5.4.5
deoxycytidine + H2O
-
654757
Bacillus subtilis
deoxyuridine + NH3
-
-
-
?
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.4.5
overexpression of wild-type enzyme and mutant enzymes R56A, R56Q and C53H/R56Q in Escherichia coli
Bacillus subtilis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
3.5.4.5
hanging drop vapour diffusion method. R56A and R56Q crystallize in the same space group as the wild-type enzyme with two subunits in the asymmetric unit, whereas C53H/R56Q can not crystallize in this crystal form but is crystallized in another space group, P3(2)21, with a full tetramer in the asymmetric unit
Bacillus subtilis
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
3.5.4.5
C53H
attempts to express and purify the mutant enzyme are unsuccessful
Bacillus subtilis
3.5.4.5
C53H/R56Q
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Because of dissociation into its inactive subunits, it is impossible to determine the kinetic parameters for the mutant enzyme
Bacillus subtilis
3.5.4.5
R56A
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Similar KM-value but decreased Vmax-value compared to wild-type enzyme
Bacillus subtilis
3.5.4.5
R56D
zinc-binding capacity of mutant enzyme is reduced
Bacillus subtilis
3.5.4.5
R56Q
mutant enzyme contains the same amount of zinc as the wild-type enzyme. Similar KM-value but decreased Vmax-value compared to wild-type enzyme
Bacillus subtilis
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.4.5
0.0075
-
deoxycytidine
pH 7.8, 25°C, mutant enzyme R56A
Bacillus subtilis
3.5.4.5
0.029
-
deoxycytidine
pH 7.8, 25°C, mutant enzyme R56Q
Bacillus subtilis
3.5.4.5
0.14
-
cytidine
pH 7.6, 25°C, mutant enzyme R56A
Bacillus subtilis
3.5.4.5
0.216
-
cytidine
pH 7.6, 25°C, wild-type enzyme
Bacillus subtilis
3.5.4.5
0.236
-
deoxycytidine
pH 7.8, 25°C, wild-type enzyme
Bacillus subtilis
3.5.4.5
0.267
-
cytidine
pH 7.6, 25°C, mutant enzyme R56Q
Bacillus subtilis
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
3.5.4.5
Zinc
wild-type enzyme and mutant enzymes R56A, R56Q and C53H/R56Q contain about 1 mol of zinc per subunit. Mutant enzyme R56D contains 0.2 mol of zinc per subunit
Bacillus subtilis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.4.5
-
Bacillus subtilis
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.4.5
cytidine + H2O
-
654757
Bacillus subtilis
uridine + NH3
-
-
-
?
3.5.4.5
deoxycytidine + H2O
-
654757
Bacillus subtilis
deoxyuridine + NH3
-
-
-
?