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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1uaq
Original Entry
Title:
Hydrolase
Compound:
Cytosine deaminase
Mutant:
No
UniProt/Swiss-Prot:
Q12178-FCY1_YEAST
EC Class:
3.5.4.1
Other CSA Entries:
Overview of all sites for 1uaq
Homologues of 1uaq
Entries for UniProt/Swiss-Prot: Q12178
Entries for EC: 3.5.4.1
Other Databases:
PDB entry: 1uaq
PDBsum entry: 1uaq
UniProt/Swiss-Prot: Q12178
IntEnz entry: 3.5.4.1
Literature Report:
Introduction:
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.
Mechanism:
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.
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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUA 64 64Sidechain
Acid/baseSubstrate
Acid/baseWater
Deprotonates the zinc-bound water to give a stronger nucleophile and protonates N3 of cytosine to generate a stronger electrophile. Deprotonates the zinc bound C4 hydroxyl of the intermediate and then protonates the departing C4 amino group. Deprotonates attacking water molecule during formation of the gem-diol intermediate. Protonates the C4-OZn oxygen and then deprotonates the C4-OH during cleavage of the C4-OZn bond.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15535715 Current protein Computer modelling
PubMed ID 12906827 Current protein Conservation of residue
PubMed ID 15535715 Current protein Structural similarity to homologue of known mechanism
PubMed ID 12637534 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 7703234 Related protein: UniProt P13652 Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
SERA 89 89Backbone carbonyl
ElectrostaticTransition state
Forms a strong hydrogen bond to the departing amino group specifically in the transition state that corresponds to cleavage of the C4-N bond.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15535715 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 15535715 Current protein Computer modelling

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 91 91Backbone amide
ElectrostaticTransition state
Forms a strong hydrogen bond to the zinc-bound oxygen specifically in the transition state the corresponds to cleavage of the C4-OZn bond in the last stages of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15535715 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 15535715 Current protein Computer modelling

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ZNA 200 0
ElectrostaticWater
Lowers pKa of nucleophilic water to allow deprotonation by Glu 64.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15535715 Current protein Computer modelling
PubMed ID 15535715 Current protein Structural similarity to homologue of known mechanism
PubMed ID 12637534 Current protein Residue is positioned appropriately (ligand position known)
References:
1
Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution.
T. P. Ko and J. J. Lin and C. Y. Hu and Y. H. Hsu and A. H. Wang and S. H. Liaw
J Biol Chem 278, (21) 19111-7, (2003).
12637534
2
The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy.
G. C. Ireton and M. E. Black and B. L. Stoddard
Structure (Camb) 11, (8) 961-72, (2003).
12906827
3
Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue.
D. C. Carlow and A. A. Smith and C. C. Yang and S. A. Short and R. Wolfenden
Biochemistry 34, (13) 4220-4, (1995).
7703234
4
Catalytic mechanism of yeast cytosine deaminase: an ONIOM computational study.
S. Sklenak and L. Yao and R. I. Cukier and H. Yan
J Am Chem Soc 126, (45) 14879-89, (2004).
15535715
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