Very short patch repair endonuclease

 

Very short patch repair protein (VSR) is an endonuclease from Escherichia coli and is a sequence specific mismatch endonuclease. It is responsible for the repair of T:G mismatches arising from the deamination of 5-methylcytosine. E. coli has three discovered systems for mismatched base pair recognition, all with different mechanisms. VSR initiates repair by hydrolysing the phosphate 5' of the mismatched T.

 

Reference Protein and Structure

Sequence
P09184 UniProt (3.1.-.-) IPR004603 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1cw0 - CRYSTAL STRUCTURE ANALYSIS OF VERY SHORT PATCH REPAIR (VSR) ENDONUCLEASE IN COMPLEX WITH A DUPLEX DNA (2.3 Å) PDBe PDBsum 1cw0
Catalytic CATH Domains
3.40.960.10 CATHdb (see all for 1cw0)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.-.-)

water
CHEBI:15377ChEBI
+
single-stranded DNA
CHEBI:9160ChEBI
→
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
hydron
CHEBI:15378ChEBI

Enzyme Mechanism

Introduction

The pKa of His 69 is raised by hydrogen bonding to Asp 97. Its raised pKa enables His 69 to act as a general base and deprotonate a water molecule. The activated water molecule then nucleophilically attacks the scissile phosphate via an SN2 mechanism, going through a trigonal-bipyramidal transition state. This negatively charged transition state is stabilised by two magnesium ions, and possibly the sidechain of His 64.

Catalytic Residues Roles

UniProt PDB* (1cw0)
Thr63 (main-C), Asp51 Thr63(62)A(D) (main-C), Asp51(50)A(D) Forms the Magnesium binding site metal ligand
Asp97 Asp97(96)A(D) Hydrogen bonds to His 69, increasing its pKa and thereby facilitating its role as a base. electrostatic stabiliser
His69 His69(68)A(D) Acts as a general base by deprotonating a water molecule, activating it for nucleophilic attack. 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

proton transfer, bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Elliott SL et al. (2005), J Mol Biol, 353, 692-703. Mechanism of the Escherichia coli DNA T:G-mismatch Endonuclease (Vsr protein) Probed with Thiophosphate-containing Oligodeoxynucleotides. DOI:10.1016/j.jmb.2005.08.048. PMID:16188275.

Step 1. His69 abstracts a proton from a water which activates it to attack the scissile phosphate via an SN2 mechanism.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp97(96)A(D) electrostatic stabiliser
Asp51(50)A(D) metal ligand
Thr63(62)A(D) (main-C) metal ligand
His69(68)A(D) proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed

Step 2. His 69 protonates the 2' oxygen.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp97(96)A(D) electrostatic stabiliser
Asp51(50)A(D) metal ligand
Thr63(62)A(D) (main-C) metal ligand
His69(68)A(D) proton donor

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His69 abstracts a proton from a water which activates it to attack the scissile phosphate via an SN2 mechanism.

Step 2. His 69 protonates the 2' oxygen.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Charity Hornby