Type II site-specific deoxyribonuclease FokI
FokI is a member an unusual class of restriction enzymes that recognise a specific DNA sequence and cleave non-specifically a short distance away from that sequence. FokI consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain.
Reference Protein and Structure
- Sequence
-
P14870
(3.1.21.4)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Planomicrobium okeanokoites (Bacteria)
- PDB
-
2fok
- STRUCTURE OF RESTRICTION ENDONUCLEASE FOKI
(2.3 Å)
- Catalytic CATH Domains
-
3.40.91.30
(see all for 2fok)
- Cofactors
- Magnesium(2+) (1)
Enzyme Mechanism
Introduction
There is little currently known about the exact mechanism of DNA cleavage. Kinetic studies have shown a cooperative second-order cleavage mechanism. Based on other enzymes that perform similar chemistry, it is assumed that the lysine residue activates the water molecule and the acidic residues form part of the metal binding site(s).
Catalytic Residues Roles
| UniProt | PDB* (2fok) | ||
| Asp454, Asp454, Asp471, Asp471 | Asp450A, Asp450A, Asp467A, Asp467A | Possibly act as magnesium binding ligands. | metal ligand |
| Lys473 | Lys469A | Possibly responsible for activating the catalytic water molecule and stabilising the reactive intermediates and transition states formed during the course of the reaction. | modifies pKa, 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
- Wah DA et al. (1997), Nature, 388, 97-100. Structure of the multimodular endonuclease FokI bound to DNA. DOI:10.1038/40446. PMID:9214510.
- Halford SE et al. (2011), Biochem Soc Trans, 39, 584-588. The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites. DOI:10.1042/BST0390584. PMID:21428944.
- Vanamee ES et al. (2007), J Mol Biol, 370, 207-212. An EM view of the FokI synaptic complex by single particle analysis. DOI:10.1016/j.jmb.2007.04.066. PMID:17524420.
- Wah DA et al. (1998), Proc Natl Acad Sci U S A, 95, 10564-10569. Structure of FokI has implications for DNA cleavage. DOI:10.1073/pnas.95.18.10564. PMID:9724743.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Asp450A | metal ligand |
| Asp467A | metal ligand |
| Lys469A | modifies pKa, electrostatic stabiliser |