Type II site-specific deoxyribonuclease, BgII

 

The type-II restriction enzymes catalyse the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. This is a large group of enzymes which require only Mg(II) and recognise specific short DNA sequences and cleave either within, or at a short specific distance from, the recognition site. This entry represents the BgII family (IPR011543) which recognises the GCCNNNNNGGC motif and cleaves after N-4.

 

Reference Protein and Structure

Sequence
O68557 UniProt (3.1.21.4) IPR011543 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis (Bacteria) Uniprot
PDB
1dmu - Crystal structure of the restriction endonuclease BglI (e.c.3.1.21.4) bound to its dna recognition sequence (2.2 Å) PDBe PDBsum 1dmu
Catalytic CATH Domains
3.40.600.20 CATHdb (see all for 1dmu)
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

Based on the active site structure, one possible mechanism is that the 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. The attacking water is activated by Lys144, it is currently unclear if Lys144 also acts as a general acid/base, or simply activates the water.

Catalytic Residues Roles

UniProt PDB* (1dmu)
Asp142 Asp142A(B) Forms part of the magnesium 1 binding site, also activates a second active site water to act as a general acid/base to protonate the O3' leaving atom. modifies pKa, metal ligand
Ile143 (main-C) Ile143A(B) (main-C) Forms part of the magnesium 1 binding site. metal ligand
Asp116 Asp116A(B) Forms part of both magnesium binding sites. metal ligand
Lys144 Lys144A(B) Activates the substrate water to act as a catalytic nulceophile, it is also responsible for stabilising the reactive intermediates. increase nucleophilicity, electrostatic stabiliser
*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. Newman M et al. (1998), EMBO J, 17, 5466-5476. Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence. DOI:10.1093/emboj/17.18.5466. PMID:9736624.
  2. Dall'Acqua W et al. (2000), Protein Sci, 9, 1-9. Substrate-assisted catalysis: Molecular basis and biological significance. DOI:10.1110/ps.9.1.1. PMID:10739241.
  3. Gormley NA et al. (2000), J Biol Chem, 275, 6928-6936. Reactions of BglI and Other Type II Restriction Endonucleases with Discontinuous Recognition Sites. DOI:10.1074/jbc.275.10.6928.

Step 1.

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Catalytic Residues Roles

Residue Roles
Asp116A(B) metal ligand
Asp142A(B) metal ligand
Ile143A(B) (main-C) metal ligand
Lys144A(B) increase nucleophilicity
Asp142A(B) modifies pKa
Lys144A(B) electrostatic stabiliser

Chemical Components

Step 1.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday