3'-5' exonuclease

 

Escherichia coli replicative DNA polymerase III, a member of family C of DNA polymerases, serves as the main DNA polymerase for E. coli. The polymerase activity is catalysed by the alpha subunit, with the epsilon subunit catalysing the 3'-5' exonuclease proofreading activity. The N-terminal domain of the epsilon subunit is the archetypal member of the DnaQ superfamily of 3'-5' exonucleases.

 

Reference Protein and Structure

Sequence
P03007 UniProt (2.7.7.7) IPR006309 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1j53 - Structure of the N-terminal Exonuclease Domain of the Epsilon Subunit of E.coli DNA Polymerase III at pH 8.5 (1.8 Å) PDBe PDBsum 1j53
Catalytic CATH Domains
3.30.420.10 CATHdb (see all for 1j53)
Cofactors
Manganese(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.11.-)

DNA polyanion
CHEBI:83828ChEBI
+
water
CHEBI:15377ChEBI
→
DNA polyanion
CHEBI:83828ChEBI

Enzyme Mechanism

Introduction

The active site contains two metal ions (A and B), which are likely to be magnesium in the native enzyme, but manganese can also be accommodated resulting in a faster reaction rate. They are coordinated to conserved acidic residues: Asp 12, Glu 14 and Asp 167. Asp 103 binds two fixed water molecules that also coordinate Metal B. The overall reaction is back-side attack of the water nucleophile on the phosphate linking the terminal nucleoside to the rest of the DNA molecule, shortening the newly replicated DNA by one base - the mechanism is common to two-metal 3'-5' exonucleases. The proposed mechanism is: Metal A activates water as nucleophile. His 162, made more basic by hydrogen bonding to Glu 61, deprotonates water as water (as hydroxide) attacks the phosphate of the scissile phosphoester bond. This phosphate group is made more electrophilic by coordination to Metal B. Metal B stabilises the negative charge on the phosphate group in the single transition state. Glu 14 deprotonates the phosphorane (abstracting the proton from what was the attacking hydroxide) triggering collapse of the intermediate. Metal B coordinates the 3' phosphate oxygen of the substrate, assisting it to leave. The products of the reaction are a dNMP and the shortened DNA molecule.

Catalytic Residues Roles

UniProt PDB* (1j53)
Glu14 Glu14A Glu 14 coordinates Metal A and may orient the attacking hydroxide. Glu 14 may also have a role in deprotonating the phosphorane intermediate, triggering collapse and product formation. hydrogen bond acceptor, metal ligand, proton acceptor, proton donor
Asp12, Asp167, Glu14 Asp12A, Asp167A, Glu14A Coordinate to the two metal ions present. metal ligand
Glu61 Glu61A Glu 61 hydrogen bonds to His 162; this may increase the basicity of His 162. increase basicity, electrostatic stabiliser, polar interaction
His162 His162A His 162 acts as a general base to deprotonate water, facilitating effective attack of hydroxide. 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, overall product formed, proton transfer, inferred reaction step, native state of enzyme regenerated

References

  1. Cisneros GA et al. (2009), J Am Chem Soc, 131, 1550-1556. Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues. DOI:10.1021/ja8082818. PMID:19119875.
  2. Johansson E et al. (2013), Cold Spring Harb Perspect Biol, 5,Replicative DNA polymerases. DOI:10.1101/cshperspect.a012799. PMID:23732474.
  3. Yang W (2011), Q Rev Biophys, 44, 1-93. Nucleases: diversity of structure, function and mechanism. DOI:10.1017/s0033583510000181. PMID:20854710.
  4. Hamdan S et al. (2002), Structure, 10, 535-546. Structural Basis for Proofreading during Replication of the Escherichia coli Chromosome. DOI:10.1016/s0969-2126(02)00738-4. PMID:11937058.

Step 1. Catalytic metal ion coordinating to the water molecule bound increases the waters nucleophilicity to attack the phosphate and promotes water deprotonation.

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Catalytic Residues Roles

Residue Roles
Glu61A electrostatic stabiliser
Asp12A metal ligand
Glu14A metal ligand
Asp167A metal ligand
Glu61A polar interaction, increase basicity
Glu14A hydrogen bond acceptor
His162A proton acceptor

Chemical Components

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

Step 2. After the phosphate ester bond has broken, Glu14 is protonated.

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Catalytic Residues Roles

Residue Roles
Asp12A metal ligand
Glu14A metal ligand
Asp167A metal ligand
Glu61A polar interaction
Glu14A proton acceptor

Chemical Components

proton transfer, overall product formed

Step 3. Water coordinated to Metal ion B protonates 3'-OH leaving group. Glu14 and His162 are deprotonated ready for another round of catalysis.

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Catalytic Residues Roles

Residue Roles
Asp12A metal ligand
Glu14A metal ligand
Asp167A metal ligand
Glu61A polar interaction
His162A proton donor
Glu14A proton donor

Chemical Components

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

Step 1. Catalytic metal ion coordinating to the water molecule bound increases the waters nucleophilicity to attack the phosphate and promotes water deprotonation.

Step 2. After the phosphate ester bond has broken, Glu14 is protonated.

Step 3. Water coordinated to Metal ion B protonates 3'-OH leaving group. Glu14 and His162 are deprotonated ready for another round of catalysis.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Morwenna Hall