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Literature summary extracted from
- Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine (2008), J. Mol. Biol., 377, 854-869.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.5.4.1 | expression of wild-type and mutant enzymes in Escherichia coli enzyme-deficient strain GIA39(DE3), genetic complementation assays, construction of mammalian expression vectors, overview | Saccharomyces cerevisiae |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 3.5.4.1 | recombinant A23L/D92E/V108I/I140L mutant bound to the transition state analogue, hanging drop vapour diffusion method, 2 days, crystals are soaked for 20 min in a mother liquor solution containing 2-hydroxypyrimidine concentrated 1.2:1 relative to protein, after soaking, the crystals are immediately transferred briefly to a cryo-buffer containing the 2-hydroxypyrimidine mother liquor plus 25% DMSO, X-ray diffraction structure determination and analysis at 2.3 A resolution, modelling and molecular replacement | Saccharomyces cerevisiae |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 3.5.4.1 | A23L/D92E/V108I/I140L | construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions | Saccharomyces cerevisiae |
| 3.5.4.1 | A23L/I140L | site-directed mutagenesis, the mutant display elevated unfolding temperatures in denaturation experiments and increased half-lives of catalytic activity at elevated temperatures, the mutant cells show an about 30% reduced sensitivity to 5-fluorouracil compared to the wild-type cells | Saccharomyces cerevisiae |
| 3.5.4.1 | A23L/M93L/V108I/I140L | construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions | Saccharomyces cerevisiae |
| 3.5.4.1 | A23L/V108I/I140L | site-directed mutagenesis, display elevated unfolding temperatures in denaturation experiments and increased half-lives of catalytic activity at elevated temperatures, the mutant cells show an about 50% reduced sensitivity to 5-fluorouracil compared to the wild-type cells | Saccharomyces cerevisiae |
| 3.5.4.1 | A23L/V108I/I140L/I98L | construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions | Saccharomyces cerevisiae |
| 3.5.4.1 | D92E | random mutagenesis, the mutation is located at the enzyme's dimer interface, the mutant shows increased thermal stability with elevated Tm values and increased activity half-life compared to the wild-type enzyme, the mutant cells show an about 30% reduced sensitivity to 5-fluorouracil compared to the wild-type cells | Saccharomyces cerevisiae |
| 3.5.4.1 | additional information | to create enzyme variants with increased activity to 5-fluorocytosine, 11 codons within the most conserved region of the enzyme, T83, L84, Y85, T86, L88, S89, D92, M93, T95, G96, and I98 are subjected to regiospecific, partially randomizing mutagenesis, Absolutely conserved residues, T87, P90, C91, and C94, within this same region, assumed to be critical for activity, are omitted from randomization, overview | Saccharomyces cerevisiae |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.5.4.1 | 0.068 | - |
5-fluorocytosine | mutant A23L/D92E/V108I/I140L | Saccharomyces cerevisiae |  |
| 3.5.4.1 | 0.083 | - |
5-fluorocytosine | wild-type enzyme | Saccharomyces cerevisiae | |
| 3.5.4.1 | 0.14 | - |
5-fluorocytosine | mutant D92E | Saccharomyces cerevisiae | |
| 3.5.4.1 | 0.15 | - |
5-fluorocytosine | mutant A23L/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 0.47 | - |
cytosine | mutant A23L/D92E/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 0.52 | - |
cytosine | mutant A23L/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 0.57 | - |
cytosine | mutant D92E | Saccharomyces cerevisiae | |
| 3.5.4.1 | 1.17 | - |
cytosine | wild-type enzyme | Saccharomyces cerevisiae | |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.5.4.1 | 5-fluorocytosine + H2O | Saccharomyces cerevisiae | inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme | 5-fluorouracil + NH3 | - |
? | |
| 3.5.4.1 | cytosine + H2O | Saccharomyces cerevisiae | the enzyme is a pyrimidine salvage pathway enzyme, inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme | uracil + NH3 | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.5.4.1 | Saccharomyces cerevisiae | Q12178 | - |
- |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.5.4.1 | 5-fluorocytosine + H2O | inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme | Saccharomyces cerevisiae | 5-fluorouracil + NH3 | - |
? | |
| 3.5.4.1 | 5-fluorocytosine + H2O | activation of the prodrug, 5-fluorouracil is an inhibitor of DNA synthesis and RNA function | Saccharomyces cerevisiae | 5-fluorouracil + NH3 | - |
? | |
| 3.5.4.1 | cytosine + H2O | - |
Saccharomyces cerevisiae | uracil + NH3 | - |
? | |
| 3.5.4.1 | cytosine + H2O | the enzyme is a pyrimidine salvage pathway enzyme, inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme | Saccharomyces cerevisiae | uracil + NH3 | - |
? | |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 3.5.4.1 | 37 | - |
in vivo assay at | Saccharomyces cerevisiae |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.5.4.1 | 9.5 | - |
cytosine | mutant A23L/D92E/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 9.5 | - |
5-fluorocytosine | mutant A23L/D92E/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 18.8 | - |
5-fluorocytosine | wild-type enzyme | Saccharomyces cerevisiae | |
| 3.5.4.1 | 29.5 | - |
cytosine | mutant D92E | Saccharomyces cerevisiae | |
| 3.5.4.1 | 29.5 | - |
5-fluorocytosine | mutant D92E | Saccharomyces cerevisiae | |
| 3.5.4.1 | 31.7 | - |
cytosine | mutant A23L/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 31.7 | - |
5-fluorocytosine | mutant A23L/V108I/I140L | Saccharomyces cerevisiae | |
| 3.5.4.1 | 170 | - |
cytosine | wild-type enzyme | Saccharomyces cerevisiae | |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 3.5.4.1 | 7.5 | - |
assay at | Saccharomyces cerevisiae |
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