Cofactors

There are no Cofactors for this Enzyme

Reaction Mechanisms

cytosine deaminase (yeast) By Reference

Cytosine deaminase catalyses the hydrolytic deamination of cytosine to uracil. It will also catalyse the deamination of the prodrug 5-fluorocytosine. The is present in bacterial and fungal cells, where it plays an important role in pyrimidine salvage, but not in mammalian cells which use cytidine deaminase instead. The bacterial and yeast cytosine deaminases are have dissimilar folds and catalytic site architectures, and have evolved independently. Yeast cytosine deaminase is however structurally related to the extensively studied bacterial cytidine deaminase. It has a very similar catalytic apparatus and is thought to use the same mechanism.




• Summary
• Step 1
• Step 2
• Step 3
• Step 4
• Step 5
• Step 6
• Step 7
• Step 8
• Step 9
• Products

The key catalytic residues are Glu 64 and a zinc ion. Glu 64 first deprotonates the zinc-bound water molecule and protonates N3 of cytosine, thus activating both nucleophile and electrophile. Attack by the water molecule on C4 then generates a tetrahedral intermediate. Generation of the tetrahedral intermediate is thought to occur in this stepwise fashion since calculations suggest that a concerted mechanism would have a much higher energy barrier. Collapse of the tetrahedral intermediate involves deprotonation of the zinc-bound C4 hydroxyl followed by cleavage of the C4-N bond with concerted protonation of the departing amino group by Glu 64.
The produced uracil is still coordinated to the zinc by O4. Calculations suggest that the energy barrier for cleavage of this bond is surprisingly high, so it is proposed that freeing of the uracil from the active site involves a gem-diol intermediate and oxygen-exchange. Formation of the gem-diol intermediate involves attack by a water molecule on C4, with concomitant deprotonation of this water molecule by Glu 64. Protonation of the the C4-O-Zn oxygen by Glu 64 is followed by cleavage of the C4-OHZn bond with simulataneous deprotonation of C4-OH by Glu 64.

Catalytic Residues

AA	Uniprot	Uniprot Resid	PDB	PDB Resid
Cys	Q12178	94	1uaq	94
His	Q12178	62	1uaq	62
Cys	Q12178	91	1uaq	91
Cys	Q12178	91	1uaq	91
Glu	Q12178	64	1uaq	64
Ser	Q12178	89	1uaq	89

Step Components

cofactor used, intermediate formation, native state of cofactor regenerated, bimolecular nucleophilic addition, rate-determining step, proton transfer, native state of enzyme regenerated, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed



Step 1.

Glu64 deprotonates a zinc bound water.



Step 2.

N3 of cytosine accepts a proton from Glu64.



Step 3.

The deprotonated water can now nucleophilicaklly attack C4 of cytosine.



Step 4.

Glu64 deprotonates the hydroxyl.



Step 5.

The deprotonation of the hydroxyl results in the initiation of an elimination from the oxyanion. This results in the cleavage of the C3 amino group which accepts a proton from Glu64 to produce ammonia.



Step 6.

Glu64 deprotonates a water so that it can attack the C3 carbon of the zinc bound uracil.



Step 7.

Glu64 protonates the oxygen bound zinc to form the gemdiol intermediate.



Step 8.

Glu64 accepts a proton from the hydroxyl which isn't coordinated to zinc on C3 which results in the cleavage of the zinc bound hydroxyl from uracil.



Step 9.

The zinc bound hydroxyl deprotonates Glu64 which regenerates the cofactor to its native state and the active site.



Products.

The products of the reaction.

View All 2 Reaction Mechanisms in M-CSA

Reaction Parameters

• Kinetic Parameters

  Organism	KM Value [mM]	Substrate	Comment
  Escherichia coli	0.004	cytosine	mutant D313N, pH 8.5, 30°C
  Saccharomyces cerevisiae	0.0000014	5-fluorocytosine	mutant E64A, pH 7.5, temperature not specified in the publication

• Temperature

  Organism	Temperature Range	Comment
  Serratia marcescens	30 - 70

• pH

  Organism	pH Range	Comment
  Salmonella enterica subsp. enterica serovar Typhimurium	6.5 - 9

Associated Proteins

Citations

• Deamination-triggered exponential signal amplification for chemiluminescent detection of cytosine deaminase at the single-cell level.
• Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase.
• Enhanced antitumor efficacy of mesenchymal stem cells expressing cytosine deaminase and 5-fluorocytosine combined with α-galactosylceramide in a colon cancer model.
• Cytosine Deaminase Base Editing to Restore COL7A1 in Dystrophic Epidermolysis Bullosa Human: Murine Skin Model.
• Genetically engineered neural stem cells expressing cytosine deaminase and interferon-beta enhanced T cell-mediated antitumor immunity against gastric cancer in a humanized mouse model.
• R-loop editing by DNA cytosine deaminase APOBEC3B determines the activity of estrogen receptor enhancers
• Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity.
• Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA
• Microbial cytosine deaminase is a programmable anticancer prodrug mediating enzyme: antibody, and gene directed enzyme prodrug therapy.
• A split cytosine deaminase architecture enables robust inducible base editing.
• Enhanced tumor inhibiting effect of 131I-BDI-1-based radioimmunotherapy and cytosine deaminase gene therapy modulated by a radio-sensitive promoter in nude mice bearing bladder cancer.