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Literature summary for 3.5.4.1 extracted from
- 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.
Application
| Application | Comment | Organism |
|---|---|---|
| medicine | cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment | Escherichia coli |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli BL21(DE3) and BL21-RIL cells | Escherichia coli |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| 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
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
| 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]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.46 | - |
cytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli |  |
| 1.65 | - |
cytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 3.16 | - |
cytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 3.76 | - |
5-fluorocytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli | |
| 4.9 | - |
cytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 6.73 | - |
5-fluorocytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 7.55 | - |
5-fluorocytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 12.69 | - |
5-fluorocytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
- |
- |
| Escherichia coli GIA39(DE3) | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| Ni-NTA column chromatography | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 5-fluorocytosine + H2O | - |
Escherichia coli | 5-fluorouracil + NH3 | - |
? | |
| 5-fluorocytosine + H2O | - |
Escherichia coli GIA39(DE3) | 5-fluorouracil + NH3 | - |
? | |
| cytosine + H2O | - |
Escherichia coli | uracil + NH3 | - |
? | |
| cytosine + H2O | - |
Escherichia coli GIA39(DE3) | uracil + NH3 | - |
? | |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.69 | - |
cytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 2.13 | - |
cytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 2.98 | - |
cytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 19.71 | - |
5-fluorocytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli | |
| 49.68 | - |
cytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli | |
| 83.68 | - |
5-fluorocytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 101.7 | - |
5-fluorocytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 115.2 | - |
5-fluorocytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.35 | - |
cytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 0.94 | - |
cytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 1.29 | - |
cytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 5.24 | - |
5-fluorocytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli | |
| 8.02 | - |
5-fluorocytosine | mutant enzyme V152A/F316C/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 12.44 | - |
5-fluorocytosine | mutant enzyme D314E/F316L/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 15.27 | - |
5-fluorocytosine | mutant enzyme V315L/F316V/D317G, pH and temperature not specified in the publication | Escherichia coli | |
| 106.8 | - |
cytosine | wild type enzyme, pH and temperature not specified in the publication | Escherichia coli | |
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