BRENDA - Enzyme Database

Sugar specificity of bacterial CMP kinases as revealed by crystal structures and mutagenesis of Escherichia coli enzyme

Bertrand, T.; Briozzo, P.; Assairi, L.; Ofiteru, A.; Bucurenci, N.; Munier-Lehmann, H.; Golinelli-Pimpaneau, B.; Barzu, O.; Gilles, A.M.; J. Mol. Biol. 315, 1099-1110 (2002)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
2.7.4.25
expressed in Escherichia coli BL21(DE3) cells
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.7.4.25
in complex with substrates CMP, dCMP, ara-CMP and 2,3-dideoxy-CMP, hanging drop vapor diffusion method, using ammonium sulfate in a 50 mM Tris-HCl buffer (pH 7.4), at 20C
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.7.4.25
D185A
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
Escherichia coli
2.7.4.25
R181M
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
Escherichia coli
2.7.4.25
S101A
the mutation reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.035
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.08
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.094
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.15
-
2',3'-dideoxy-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.24
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4; mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.46
-
2',3'-dideoxy-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.53
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.54
-
2',3'-dideoxy-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.65
-
2',3'-dideoxy-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.79
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.4.25
Mg2+
required for activity
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.4.25
ATP + CMP
Escherichia coli
-
ADP + CDP
-
-
?
2.7.4.25
ATP + dCMP
Escherichia coli
Escherichia coli CMPK phosphorylates dCMP nearly as well as it does CMP
ADP + dCDP
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.7.4.25
Escherichia coli
A0A140N8S7
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.7.4.25
Ni-NTA column chromatography
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.4.25
ATP + 2',3'-dideoxy-CMP
2,3-dideoxy-CMP is a poor substrate
712736
Escherichia coli
ADP + ?
-
-
-
?
2.7.4.25
ATP + ara-CMP
ara-CMP is a poor substrate
712736
Escherichia coli
ADP + ara-CDP
-
-
-
?
2.7.4.25
ATP + CMP
-
712736
Escherichia coli
ADP + CDP
-
-
-
?
2.7.4.25
ATP + dCMP
Escherichia coli CMPK phosphorylates dCMP nearly as well as it does CMP
712736
Escherichia coli
ADP + dCDP
-
-
-
?
2.7.4.25
ATP + UMP
bacterial CMP kinases phosphorylate UMP with very low rates
712736
Escherichia coli
ADP + UDP
-
-
-
?
Temperature Stability [C]
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
2.7.4.25
52
-
the melting temperature of the wild type enzyme is at 52C
Escherichia coli
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.0083
-
2',3'-dideoxy-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.047
-
2',3'-dideoxy-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.071
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.08
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.085
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.12
-
2',3'-dideoxy-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.26
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.45
-
dCMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.65
-
2',3'-dideoxy-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1.36
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1.38
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
56
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
103
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
109
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
2.7.4.25
ATP
-
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.4.25
expressed in Escherichia coli BL21(DE3) cells
Escherichia coli
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
2.7.4.25
ATP
-
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.7.4.25
in complex with substrates CMP, dCMP, ara-CMP and 2,3-dideoxy-CMP, hanging drop vapor diffusion method, using ammonium sulfate in a 50 mM Tris-HCl buffer (pH 7.4), at 20C
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.7.4.25
D185A
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
Escherichia coli
2.7.4.25
R181M
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
Escherichia coli
2.7.4.25
S101A
the mutation reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.035
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.08
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.094
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.15
-
2',3'-dideoxy-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.24
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4; mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.46
-
2',3'-dideoxy-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.53
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.54
-
2',3'-dideoxy-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.65
-
2',3'-dideoxy-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.79
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.4.25
Mg2+
required for activity
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.4.25
ATP + CMP
Escherichia coli
-
ADP + CDP
-
-
?
2.7.4.25
ATP + dCMP
Escherichia coli
Escherichia coli CMPK phosphorylates dCMP nearly as well as it does CMP
ADP + dCDP
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.4.25
Ni-NTA column chromatography
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.4.25
ATP + 2',3'-dideoxy-CMP
2,3-dideoxy-CMP is a poor substrate
712736
Escherichia coli
ADP + ?
-
-
-
?
2.7.4.25
ATP + ara-CMP
ara-CMP is a poor substrate
712736
Escherichia coli
ADP + ara-CDP
-
-
-
?
2.7.4.25
ATP + CMP
-
712736
Escherichia coli
ADP + CDP
-
-
-
?
2.7.4.25
ATP + dCMP
Escherichia coli CMPK phosphorylates dCMP nearly as well as it does CMP
712736
Escherichia coli
ADP + dCDP
-
-
-
?
2.7.4.25
ATP + UMP
bacterial CMP kinases phosphorylate UMP with very low rates
712736
Escherichia coli
ADP + UDP
-
-
-
?
Temperature Stability [C] (protein specific)
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
2.7.4.25
52
-
the melting temperature of the wild type enzyme is at 52C
Escherichia coli
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.0083
-
2',3'-dideoxy-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.047
-
2',3'-dideoxy-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.071
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.08
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.085
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.12
-
2',3'-dideoxy-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.19
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.26
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.45
-
dCMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.47
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
0.65
-
2',3'-dideoxy-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1.36
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
1.38
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli
2.7.4.25
56
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
103
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
2.7.4.25
109
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.083
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
0.3
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
0.54
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1.7
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1.9
-
dCMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
6.1
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
7.4
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
7.5
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
105
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
697
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1160
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
2940
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
2.7.4.25
0.083
-
ara-CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
0.3
-
dCMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
0.54
-
CMP
mutant enzyme D185A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1.7
-
ara-CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1.9
-
dCMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
6.1
-
dCMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
7.4
-
CMP
mutant enzyme R181M, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
7.5
-
ara-CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
105
-
ara-CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
697
-
CMP
mutant enzyme S101A, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
1160
-
dCMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12
2.7.4.25
2940
-
CMP
wild type enzyme, at 30C and pH 7.4
Escherichia coli K-12