Crossover junction endodeoxyribonuclease

 

Endonuclease VII from Bacteriophage t4 is a junction-resolving enzyme. It has a broad substrate specificity, and recognises a variety of branched DNA structures and other structural perturbations in DNA such as Y-junctions, heteroduplex loops and abasic sites.

 

Reference Protein and Structure

Sequence
P13340 UniProt (3.1.-.-) IPR004211 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T4 (Virus) Uniprot
PDB
1en7 - ENDONUCLEASE VII (ENDOVII) FROM PHAGE T4 (2.4 Å) PDBe PDBsum 1en7
Catalytic CATH Domains
3.40.1800.10 CATHdb 1.10.720.10 CATHdb (see all for 1en7)
Cofactors
Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.22.4)

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: Hje endonuclease, Holliday junction endonuclease CCE1, Holliday junction resolvase, Holliday junction-cleaving endonuclease, Holliday junction-resolving endoribonuclease, RusA Holliday junction resolvase, RusA endonuclease, RuvC endonuclease, SpCCe1 Holliday junction resolvase, Crossover junction endoribonuclease, Cruciform-cutting endonuclease, Endo X3, Endonuclease RuvC, Endonuclease VII, Endonuclease X3, Resolving enzyme CCE1, Holliday junction nuclease,

Enzyme Mechanism

Introduction

This enzyme catalyses a one step reaction which proceeds through a pentavalent transition state. His 41 acts as a general base to deprotonate water, activating it for nucleophilic attack on the phosphorus atom. This causes the P-O3' bond to break, and at the same time, His 105' protonates the O3' atom of the leaving group. Ca2+ (and possibly His 43) stabilise the pentavalent transition state.

Catalytic Residues Roles

UniProt PDB* (1en7)
His43, Glu65 His43A, Glu65A Stabilises charge on phosphate group in transition state. hydrogen bond donor, electrostatic stabiliser
His41 His41A His 41 acts as a general base, deprotonating a water molecule and activating it for nucleophilic attack on the phosphorus atom. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His105 His105B His 105 acts as a general acid by protonating the O3' atom on the leaving group as it leaves. 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

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

References

  1. Raaijmakers H et al. (2001), J Mol Biol, 308, 311-323. Conformational flexibility in T4 endonuclease VII revealed by crystallography: implications for substrate binding and cleavage1 1Edited by K. Morikawa. DOI:10.1006/jmbi.2001.4592. PMID:11327769.
  2. Raaijmakers H et al. (1999), EMBO J, 18, 1447-1458. X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture. DOI:10.1093/emboj/18.6.1447. PMID:10075917.

Step 1. His41 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 His105B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105B hydrogen bond donor
His41A hydrogen bond acceptor
His43A hydrogen bond donor, electrostatic stabiliser
Glu65A hydrogen bond donor, electrostatic stabiliser
Asp40A metal ligand
Asn62A metal ligand
His41A proton acceptor
His105B proton donor

Chemical Components

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

Step 2. His105B deprotonates His41.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105B hydrogen bond acceptor
His41A hydrogen bond donor
His105B proton acceptor
His41A proton donor

Chemical Components

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

Step 1. His41 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 His105B.

Step 2. His105B deprotonates His41.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Ellie Wright, James Torrance, James Willey