Type II site-specific deoxyribonuclease, Cfr10I/Bse634I
Type II restriction endonucleases (EC:18.104.22.168) 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
- P56200 (22.214.171.124) (Sequence Homologues) (PDB Homologues)
- Biological species
Citrobacter freundii (Bacteria)
- 1cfr - CRYSTAL STRUCTURE OF CITROBACTER FREUNDII RESTRICTION ENDONUCLEASE CFR10I AT 2.15 ANGSTROMS RESOLUTION. (2.15 Å)
- Catalytic CATH Domains
- 126.96.36.199 (see all for 1cfr)
- Magnesium(2+) (2)
Enzyme Reaction (EC:188.8.131.52)
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
|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|
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.