Type II site-specific deoxyribonuclease, Cfr10I/Bse634I

 

Type II restriction endonucleases (EC:3.1.21.4) are components of prokaryotic DNA restriction-modification mechanisms that protect the organism against invading foreign DNA. These site-specific deoxyribonucleases catalyse the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.

This entry represents Cfr10I and Bse634I restriction endonucleases (IPR012415). They exhibit a conserved tetrameric architecture with two dimers arranged back-to-back with their putative DNA-binding clefts facing opposite directions. These clefts are formed between two monomers that interact, mainly via hydrophobic interactions supported by a few hydrogen bonds, to form a U-shaped dimer. Each monomer is folded to form a compact alpha-beta structure, whose core is made up of a five-stranded mixed beta-sheet. The monomer may be split into separate N-terminal and C-terminal subdomains at a hinge located in helix alpha3. Both Cfr10I and Bse634I recognise the double-stranded sequence RCCGGY and cleave after the purine R [PMID: 8568865].

 

Reference Protein and Structure

Sequence
P56200 UniProt (3.1.21.4) IPR012415 (Sequence Homologues) (PDB Homologues)
Biological species
Citrobacter freundii (Bacteria) Uniprot
PDB
1cfr - CRYSTAL STRUCTURE OF CITROBACTER FREUNDII RESTRICTION ENDONUCLEASE CFR10I AT 2.15 ANGSTROMS RESOLUTION. (2.15 Å) PDBe PDBsum 1cfr
Catalytic CATH Domains
3.40.91.10 CATHdb (see all for 1cfr)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.21.4)

single-stranded DNA
CHEBI:9160ChEBI
+
water
CHEBI:15377ChEBI
→
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Type II restriction enzyme,

Enzyme Mechanism

Introduction

There appears to still be much discussion on the exact mechanism of these enzymes, with a review from 2008 suggesting that they may work through many different mechanisms. Especially as there appears to be no set number of metal ions in the active site; some crystal structures show no divalent metal cations, some one, and others two.

However, it appears that the general two-metal mechanism is favoured. In this mechanism a nucleophilic water is bound between the two divalent metal cations, and activated to form a hydroxide ion. This ion initiates a nucleophilic attack on the phosphorous atom. The pentavalent intermediate collapses and abstracts a proton from another water bound at one of the metal ions.

Catalytic Residues Roles

UniProt PDB* (1cfr)
Lys190 Lys190A Likely to be involved in the stabilisation of the doubly charged pentacoordinate transition state. metal ligand, electrostatic stabiliser
Glu71, Asp134 Glu71A, Asp134A Forms part of the Magnesium 2 binding site. metal ligand
Glu204 Glu204A Thought to act as a general acid/base. proton shuttle (general acid/base), metal ligand
Asp134, Glu204 Asp134A, Glu204A Forms part of the Magnesium 1 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. Xie F et al. (2010), J Inorg Biochem, 104, 665-672. Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study. DOI:10.1016/j.jinorgbio.2010.02.008. PMID:20347155.
  2. Manakova E et al. (2012), Nucleic Acids Res, 40, 6741-6751. Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease. DOI:10.1093/nar/gks300. PMID:22495930.
  3. Dupureur CM (2008), Curr Opin Chem Biol, 12, 250-255. Roles of metal ions in nucleases. DOI:10.1016/j.cbpa.2008.01.012. PMID:18261473.
  4. Grazulis S (2002), Nucleic Acids Res, 30, 876-885. Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence. DOI:10.1093/nar/30.4.876.
  5. Skirgaila R et al. (1998), J Mol Biol, 279, 473-481. Structure-based redesign of the Catalytic/Metal binding site of Cfr 10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues. DOI:10.1006/jmbi.1998.1803. PMID:9642051.
  6. Bozic D et al. (1996), J Mol Biol, 255, 176-186. Crystal Structure ofCitrobacter freundiiRestriction EndonucleaseCfr10I at 2.15 Å Resolution. DOI:10.1006/jmbi.1996.0015. PMID:8568865.

Step 1.

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Catalytic Residues Roles

Residue Roles
Glu71A metal ligand
Asp134A metal ligand
Lys190A electrostatic stabiliser
Glu204A metal ligand
Lys190A metal ligand
Glu204A proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Gemma L. Holliday, Craig Porter