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Literature summary extracted from

  • Fuchita, M.; Ardiani, A.; Zhao, L.; Serve, K.; Stoddard, B.L.; Black, M.E.
    Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo (2009), Cancer Res., 69, 4791-4799.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
3.5.4.1 medicine cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.4.1 expressed in Escherichia coli BL21(DE3) and BL21-RIL cells Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.4.1 the wild type enzyme in complex with the mechanism-based inhibitors 4-(R)-hydroxyl-3,4-dihydropyrimidine or 5-fluoro-4-(S)-hydroxyl-3,4-dihydropyrimidine and the mutant enzyme V152A/F316C/D317G are crystallized by the sitting drop vapor diffusion method, using 10-15% (w/v) PEG 6K, 200 mM MgCl2 and 100 mM HEPES (pH 7-8) Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
3.5.4.1 D314E/F316L/D317G the mutant displays 9% substrate specificity towards cytosine and 1820% substrate specificity towards 5-fluorocytosine compared to the wild type enzyme Escherichia coli
3.5.4.1 V152A/F316C/D317G the mutant displays 4% substrate specificity towards cytosine and 1920% substrate specificity towards 5-fluorocytosine compared to the wild type enzyme Escherichia coli
3.5.4.1 V315L/F316V/D317G the mutant displays 6% substrate specificity towards cytosine and 1880% substrate specificity towards 5-fluorocytosine compared to the wild type enzyme Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.4.1 0.46
-
 cytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 1.65
-
 cytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 3.16
-
 cytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 3.76
-
 5-fluorocytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 4.9
-
 cytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 6.73
-
 5-fluorocytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 7.55
-
 5-fluorocytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 12.69
-
 5-fluorocytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli

Organism

EC Number Organism UniProt Comment Textmining
3.5.4.1 Escherichia coli
-
-
-
3.5.4.1 Escherichia coli GIA39(DE3)
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.4.1 Ni-NTA column chromatography Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.4.1 5-fluorocytosine + H2O
-
 Escherichia coli 5-fluorouracil + NH3
-
 ?
3.5.4.1 5-fluorocytosine + H2O
-
 Escherichia coli GIA39(DE3) 5-fluorouracil + NH3
-
 ?
3.5.4.1 cytosine + H2O
-
 Escherichia coli uracil + NH3
-
 ?
3.5.4.1 cytosine + H2O
-
 Escherichia coli GIA39(DE3) uracil + NH3
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.4.1 1.69
-
 cytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 2.13
-
 cytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 2.98
-
 cytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 19.71
-
 5-fluorocytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 49.68
-
 cytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 83.68
-
 5-fluorocytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 101.7
-
 5-fluorocytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 115.2
-
 5-fluorocytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.4.1 0.35
-
 cytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 0.94
-
 cytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 1.29
-
 cytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 5.24
-
 5-fluorocytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 8.02
-
 5-fluorocytosine mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 12.44
-
 5-fluorocytosine mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 15.27
-
 5-fluorocytosine mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication Escherichia coli
3.5.4.1 106.8
-
 cytosine wild type enzyme, pH and temperature not specified in the publication Escherichia coli