M-CSA Mechanism and Catalytic Site Atlas

Deoxyribonuclease (pyrimidine dimer)

Endonuclease V removes pyrimidine dimers in DNA induced by UV radiation. The enzyme first associates with the DNA and locates the damaged site by a scanning mechanism. It catalyses 2 reactions to remove DNA pyrimidine dimers: (1)cleavage of glycosidic bond by pyrimidine dimer glycosylase activity, which hydrolyses the N-glycosyl bond of the 5'-thymidine in the thymine dimer site and (2)incision of the phosphodiester bond at the apyrimidinic site via a beta-elimination reaction by the apurinic/apyrimidinic(AP) endonuclease activity.

Reference Protein and Structure

Sequence: P04418 (3.2.2.17, 4.2.99.18) (Sequence Homologues) (PDB Homologues)
Biological species: Enterobacteria phage T4 (Virus)
PDB: 1vas - ATOMIC MODEL OF A PYRIMIDINE DIMER SPECIFIC EXCISION REPAIR ENZYME COMPLEXED WITH A DNA SUBSTRATE: STRUCTURAL BASIS FOR DAMAGED DNA RECOGNITION (2.75 Å)
Catalytic CATH Domains: 1.10.440.10 (see all for 1vas)

Click To Show Structure

Enzyme Reaction (EC:3.1.25.1)

water

CHEBI:15377

cyclobutadithymidine residue

CHEBI:65287

→

cyclobutane pyrimidine dimer 5'-phosphate residue

CHEBI:137035

DNA 3'-a,b-unsaturated aldehyde

CHEBI:137036

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: T4 endonuclease V, Bacteriophage T4 endodeoxyribonuclease V, Endodeoxyribonuclease (pyrimidine dimer),

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

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

Introduction

The glycosylase step proceeds by an SN2 mechanism. The neutral N-terminus(Thr2) performs a nucleophilic attack on ribose C1' of the 5'-thymine of the dimer. The susceptibility of C1' to nucleophilic attack is enhanced by a hydrogen bond between Glu23 and O4' of the ribose. The transition state is stabilised by the hydrogen bond as well. The positively charged Arg22 and Arg26 provides favourable electrostatic interactions to the transition state and hence stabilising it. Glu23 protonates ribose O4', leading to the opening of the ribose ring and the formation of the protonated Schiff base, the product of the glycosylase reaction, which is stabilised by Glu23.

The AP endonuclease step proceeds via a beta-elimination mechanism. Glu23 acts as a base to remove the pro-S 2'-hydrogen of the opened ribose, leading to the cleavage of the phosphodiester bond. It is followed by the hydrolysis of the Schiff base by a water molecule, leading to the dissociation of the enzyme and the formation of an alpha-beta-unsaturated aldehyde.

Catalytic Residues Roles

UniProt	PDB* (1vas)
Thr2 (N-term)	Thr2(1)A(C) (N-term)	The N-terminal of Thr 2 acts as a nucleophile to attack the ribose C1' of the 5'-thymine of the dimer.	covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, proton acceptor, proton donor, nucleophile, electron pair acceptor, electron pair donor
Arg22	Arg22(21)A(C)	Stabilises the transition state of the glycosylase reaction by forming favourable electrostatic interaction.	hydrogen bond donor, electrostatic stabiliser
Arg26	Arg26(25)A(C)	Stabilises the transition state in the glycosylase reaction by providing favourable electrostatic interaction.	hydrogen bond donor, electrostatic stabiliser
Glu23	Gln23(22)A(C)	In the glycosylase reaction, it hydrogen bonds to ribose O4' to increase the electrophilicity of ribose C1' to enhance the nucleophilic attack of Thr 2 on C1' The hydrogen bond stabilises the transition state as well. It protonates the ribose O4', leading to the opening of the ribose ring and the formation of the Schiff base, which is stabilised again by Glu 23. In the AP endonuclease reaction, it acts as a base to remove the pro-S 2'-hydrogen of the opened ribose, leading to the cleavage of the phosphodiester bond.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor

*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

bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, proton relay, unimolecular elimination by the conjugate base, decyclisation, schiff base formed, assisted tautomerisation (not keto-enol), intermediate collapse, overall product formed, bimolecular nucleophilic addition, enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated

References

Fuxreiter M et al. (1999), Biochemistry, 38, 9577-9589. Role of Active Site Residues in the Glycosylase Step of T4 Endonuclease V. Computer Simulation Studies on Ionization States†. DOI:10.1021/bi9901937. PMID:10423235.
Golan G et al. (2006), J Mol Biol, 362, 241-258. Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA. DOI:10.1016/j.jmb.2006.06.059. PMID:16916523.
Vassylyev DG et al. (1995), Cell, 83, 773-782. Atomic model of a pyrimidine dimer excision repair enzyme complexed with a dna substrate: Structural basis for damaged DNA recognition. DOI:10.1016/0092-8674(95)90190-6. PMID:8521494.
Schrock RD 3rd et al. (1993), J Biol Chem, 268, 880-886. Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. PMID:8419366.
Doi T et al. (1992), Proc Natl Acad Sci U S A, 89, 9420-9424. Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V. DOI:10.1073/pnas.89.20.9420. PMID:1409651.
Morikawa K et al. (1992), Science, 256, 523-526. X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer. DOI:10.1126/science.1575827. PMID:1575827.

Step 1. The N-terminus of the enzyme, Thr2, attacks the C1 carbon of the deoxyribose ring at the 5' end of the DNA molecule in a nucleophilic substitution, cleaving the bond to the damaged base

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg22(21)A(C)	hydrogen bond donor, electrostatic stabiliser
Gln23(22)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor, electrostatic stabiliser
Thr2(1)A(C) (N-term)	nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 2. The oxyanion initiates an isomerisation which causes the nitrogen of the base to deprotonate water, which in turn deprotonates the N-terminal Thr2.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached, hydrogen bond donor
Arg22(21)A(C)	hydrogen bond donor, electrostatic stabiliser
Gln23(22)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor, electrostatic stabiliser
Thr2(1)A(C) (N-term)	proton donor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 3. The lone pair on the N-terminal Thr2 initiates an elimination, cleaving the C1-O bond of the deoxyribose ring, which in turn deprotonates Glu23

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached
Arg22(21)A(C)	hydrogen bond donor
Gln23(22)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor
Gln23(22)A(C)	proton donor
Thr2(1)A(C) (N-term)	electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, decyclisation, intermediate formation, schiff base formed

Step 4. Glu23 deprotonates the C2 of the deoxyribose ring, causing a double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached
Arg22(21)A(C)	hydrogen bond donor
Gln23(22)A(C)	hydrogen bond acceptor
Arg26(25)A(C)	hydrogen bond donor
Gln23(22)A(C)	proton acceptor
Thr2(1)A(C) (N-term)	electron pair acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), intermediate formation

Step 5. The lone pair on the N-terminal Thr2 initiates an elimination of the phosphate, which in turn deprotonates Glu23

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached
Arg22(21)A(C)	hydrogen bond donor
Gln23(22)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor
Gln23(22)A(C)	proton donor
Thr2(1)A(C) (N-term)	electron pair donor

Chemical Components

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

Step 6. Glu23 deprotonates water, which attacks the C1 bound to the N-terminal Thr2 in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached
Arg22(21)A(C)	hydrogen bond donor
Gln23(22)A(C)	hydrogen bond acceptor
Arg26(25)A(C)	hydrogen bond donor
Gln23(22)A(C)	proton acceptor
Thr2(1)A(C) (N-term)	electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 7. The N-terminal Thr2 deprotonates the newly formed alcohol group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr2(1)A(C) (N-term)	covalently attached, hydrogen bond acceptor
Arg22(21)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor
Thr2(1)A(C) (N-term)	proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 8. The oxyanion initiates an elimination of the N-terminal Thr2, regenerating the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg22(21)A(C)	hydrogen bond donor
Arg26(25)A(C)	hydrogen bond donor
Thr2(1)A(C) (N-term)	nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate terminated, overall product formed, native state of enzyme regenerated