M-CSA Mechanism and Catalytic Site Atlas

Cytosine deaminase (yeast)

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.

Reference Protein and Structure

Sequence: Q12178 (3.5.4.1) (Sequence Homologues) (PDB Homologues)
Biological species: Saccharomyces cerevisiae S288c (Baker's yeast)
PDB: 1uaq - The crystal structure of yeast cytosine deaminase (1.6 Å)
Catalytic CATH Domains: 3.40.140.10 (see all for 1uaq)
Cofactors: Zinc(2+) (1)

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Enzyme Reaction (EC:3.5.4.1)

cytosine

CHEBI:16040

water

CHEBI:15377

hydron

CHEBI:15378

→

ammonium

CHEBI:28938

uracil

CHEBI:17568

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: Isocytosine deaminase, Cytosine aminohydrolase,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

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

Introduction

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 Roles

UniProt	PDB* (1uaq)
Cys94, His62, Cys91	Cys94A, His62A, Cys91A	Form Zinc coordination sphere	metal ligand
Cys91 (main-N)	Cys91A (main-N)	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.	metal ligand, electrostatic stabiliser
Glu64	Glu64A	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.	proton acceptor, proton donor
Ser89 (main-C)	Ser89A (main-C)	Forms a strong hydrogen bond to the departing amino group specifically in the transition state that corresponds to cleavage of the C4-N bond.	electrostatic stabiliser

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

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

References

Ko TP et al. (2003), J Biol Chem, 278, 19111-19117. Crystal Structure of Yeast Cytosine Deaminase: INSIGHTS INTO ENZYME MECHANISM AND EVOLUTION. DOI:10.1074/jbc.m300874200. PMID:12637534.
Zhao Y et al. (2017), Biochim Biophys Acta, 1865, 1020-1029. Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study. DOI:10.1016/j.bbapap.2017.05.001. PMID:28478051.
Zhang X et al. (2016), J Comput Chem, 37, 1163-1174. Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD). DOI:10.1002/jcc.24306. PMID:26813441.
Sklenak S et al. (2004), J Am Chem Soc, 126, 14879-14889. Catalytic Mechanism of Yeast Cytosine Deaminase: An ONIOM Computational Study. DOI:10.1021/ja046462k. PMID:15535715.
Ireton GC et al. (2003), Structure, 11, 961-972. The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy. PMID:12906827.
Carlow DC et al. (1995), Biochemistry, 34, 4220-4224. Profound contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: Mutation and rescue. DOI:10.1021/bi00013a010. PMID:7703234.

Step 1. Glu64 deprotonates a zinc bound water.

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Catalytic Residues Roles

Residue	Roles
Cys91A (main-N)	electrostatic stabiliser
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	metal ligand
His62A	metal ligand
Cys94A	metal ligand
Glu64A	proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 2. N3 of cytosine accepts a proton from Glu64.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. The deprotonated water can now nucleophilicaklly attack C4 of cytosine.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, cofactor used

Step 4. Glu64 deprotonates the hydroxyl.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton acceptor

Chemical Components

proton transfer, intermediate formation

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.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed

Step 6. Glu64 deprotonates a water so that it can attack the C3 carbon of the zinc bound uracil.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, rate-determining step

Step 7. Glu64 protonates the oxygen bound zinc to form the gemdiol intermediate.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton donor

Chemical Components

proton transfer, intermediate formation

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.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

Step 9. The zinc bound hydroxyl deprotonates Glu64 which regenerates the cofactor to its native state and the active site.

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Catalytic Residues Roles

Residue	Roles
Ser89A (main-C)	electrostatic stabiliser
Cys91A (main-N)	electrostatic stabiliser
His62A	metal ligand
Cys91A	metal ligand
Cys94A	metal ligand
Glu64A	proton donor

Chemical Components

proton transfer, native state of cofactor regenerated, native state of enzyme regenerated

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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 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.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby