Type II site-specific deoxyribonuclease PvuII

 

Catalyses the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. This class recognises the double-stranded sequence CAGCTG and cleaves after G-3.

 

Reference Protein and Structure

Sequence
P23657 UniProt (3.1.21.4) IPR015306 (Sequence Homologues) (PDB Homologues)
Biological species
Proteus hauseri (Bacteria) Uniprot
PDB
1pvi - STRUCTURE OF PVUII ENDONUCLEASE WITH COGNATE DNA (2.6 Å) PDBe PDBsum 1pvi
Catalytic CATH Domains
3.40.210.10 CATHdb (see all for 1pvi)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.21.4)

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
Alternative enzyme names: Type II restriction enzyme,

Enzyme Mechanism

Introduction

This restriction enzyme functions with two metal ions. Mg(II) ion at site 1 could help to activate the attacking water molecule, a Mg(II) at site 2 helps to stabilise the negative charge on the 3' oxyanion leaving group, and both ions are involved in stabilising the pentacovalent transition state. Lys70 is thought to act as a general acid/base.

Catalytic Residues Roles

UniProt PDB* (1pvi)
Lys70 Lys70A(C) Acts as a general acid/base. proton shuttle (general acid/base)
Asp58 Asp58A(C) Acts as a bidentate ligand between the to magnesium ions. metal ligand
Glu68 Glu68A(C) Forms part of the Mg1 binding site. metal ligand
*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

References

  1. Spyridaki A et al. (2003), J Mol Biol, 331, 395-406. Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII. PMID:12888347.
  2. Pingoud A et al. (2001), Nucleic Acids Res, 29, 3705-3727. Structure and function of type II restriction endonucleases. PMID:11557805.
  3. Horton JR et al. (2000), J Mol Biol, 300, 1049-1056. PvuII endonuclease contains two calcium ions in active sites. DOI:10.1006/jmbi.2000.3938. PMID:10903853.
  4. Horton JR et al. (1998), J Mol Biol, 284, 1491-1504. Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis. DOI:10.1006/jmbi.1998.2269. PMID:9878366.
  5. Cheng X et al. (1994), EMBO J, 13, 3927-3935. Structure of PvuII endonuclease with cognate DNA. PMID:8076590.

Step 1.

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Catalytic Residues Roles

Residue Roles
Asp58A(C) metal ligand
Glu68A(C) metal ligand
Lys70A(C) proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday