Exodeoxyribonuclease III

 

Exonuclease III from Escherichia coli is a multifunctional enzyme which is able to cleave DNA in order to facilitate the removal of a particular base to create an AP site. This process occurs during replication, allowing incorrectly placed base pairs to be removed and replaced by the correct one. The efficiency of this process is essential to protect Escherichia coli from the damaging effects of mutations, and enables the fidelity of DNA polymerase activity to be increased by over 1000 times.

 

Reference Protein and Structure

Sequence
P09030 UniProt (3.1.11.2) IPR004808 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ako - EXONUCLEASE III FROM ESCHERICHIA COLI (1.7 Å) PDBe PDBsum 1ako
Catalytic CATH Domains
3.60.10.10 CATHdb (see all for 1ako)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.11.2)

water
CHEBI:15377ChEBI
+
single-stranded DNA
CHEBI:9160ChEBI
→
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: E. coli exonuclease III, Escherichia coli exonuclease III, Endoribonuclease III, Exonuclease III, E.coli exonuclease III,

Enzyme Mechanism

Introduction

The phosphodiester bond is cleaved at the 3' end to leave a free 3' OH group and a phosphorylated 5' end. This occurs through the nucleophilic attack of a water molecule, activated by deprotonation from His 259 which is itself primed through hydrogen bonding contacts with Asp 229. The bond formed between the water molecule and the phosphorous atom allows a pentavalent phosphate transition state to develop, stabilised by electrostatic contacts from a Mg(II) ion at the active site. This collapses to release the products and cleave the DNA backbone, following protonation of the 3' by Asp 151's side chain, which is primed by contacts with Asn 7 and Asn 153 to ensure that it is protonated at physiological pH.

Catalytic Residues Roles

UniProt PDB* (1ako)
Asn153 Asn153A Forms contacts to ensure that Asp 151 is protonated at physiological pH and can act as an acid. Forms part of the magnesium 2 binding site. activator, hydrogen bond donor, metal ligand, electrostatic stabiliser
Asp229 Asp229A Acts to prime His 259 to allow it to act as an acid/base at physiological pH. activator, hydrogen bond acceptor, electrostatic stabiliser
Asn7 Asn7A Forms contacts to Asp 151 to ensure that it is protonated and can act as an acid. activator, hydrogen bond donor, electrostatic stabiliser
Asp258, Glu34 Asp258A, Glu34A Forms part of the magnesium 1 binding site. metal ligand
His259 His259A Deprotonates the water molecule that attacks the electrophilic phosphorous atom to result in the cleavage of the phosphodiester bond. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp151 Asp151A Acts as acid to protonate the leaving group. Forms part of the magnesium 2 binding site. hydrogen bond acceptor, hydrogen bond donor, metal ligand, 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, hydrolysis, native state of enzyme regenerated, inferred reaction step

References

  1. Mol CD et al. (1995), Nature, 374, 381-386. Structure and function of the multifunctional DNA-repair enzyme exonuclease III. DOI:10.1038/374381a0. PMID:7885481.
  2. Black CB et al. (1997), Eur J Biochem, 243, 684-689. Inert Chromium and Cobalt Complexes as Probes of Magnesium-Dependent Enzymes. Evaluation of the Mechanistic Role of the Essential Metal Cofactor in Escherichia Coli Exonuclease III. DOI:10.1111/j.1432-1033.1997.00684.x. PMID:9057832.
  3. Kuo CF et al. (1994), Ann N Y Acad Sci, 726, 223-235. Structure and Function of the DNA Repair Enzyme Exonuclease III from E. Coli. DOI:10.1111/j.1749-6632.1994.tb52820.x. PMID:8092679.

Step 1. His259 deprotonates water, which attacks the phosphate of DNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the DNA molecule reprotonates from Asp151.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn7A hydrogen bond donor, electrostatic stabiliser
Asp151A hydrogen bond acceptor, hydrogen bond donor
Asn153A hydrogen bond donor, electrostatic stabiliser
Asp229A hydrogen bond acceptor, activator
His259A hydrogen bond acceptor, hydrogen bond donor
Glu34A metal ligand
Asp258A metal ligand
Asp151A metal ligand
Asn153A metal ligand
His259A proton acceptor
Asp151A proton donor

Chemical Components

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

Step 2. Asp151 deprotonates His259.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn7A hydrogen bond donor, activator
Asp151A hydrogen bond acceptor
Asn153A hydrogen bond donor, activator
Asp229A hydrogen bond acceptor, electrostatic stabiliser
His259A hydrogen bond donor
Glu34A metal ligand
Asp258A metal ligand
Asp151A metal ligand
Asn153A metal ligand
His259A proton donor
Asp151A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. His259 deprotonates water, which attacks the phosphate of DNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the DNA molecule reprotonates from Asp151.

Step 2. Asp151 deprotonates His259.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Peter Sarkies, James Willey