PDBsum entry 2ge5

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protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
Protein chains
219 a.a.
Waters ×45
PDB id:
Name: Hydrolase/DNA
Title: Ecorv restriction endonucleasE C-terminal deletion mutant/gatatc/ca2+
Structure: 5'-d( Ap Ap Ap Gp Ap Tp Ap Tp Cp Tp T)-3'. Chain: c, d. Engineered: yes. Type ii restriction enzyme ecorv. Chain: a, b. Fragment: residues 1-219. Synonym: endonuclease ecorv, r.Ecorv. Engineered: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Gene: ecorvr. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
2.40Å     R-factor:   0.223     R-free:   0.284
Authors: D.A.Hiller,J.J.Perona
Key ref:
D.A.Hiller and J.J.Perona (2006). Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage. Biochemistry, 45, 11453-11463. PubMed id: 16981705 DOI: 10.1021/bi0606400
17-Mar-06     Release date:   04-Jul-06    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
245 a.a.
219 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Type Ii site-specific deoxyribonuclease.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
      Cofactor: Mg(2+)
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biochemical function     DNA binding     2 terms  


DOI no: 10.1021/bi0606400 Biochemistry 45:11453-11463 (2006)
PubMed id: 16981705  
Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage.
D.A.Hiller, J.J.Perona.
The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19658127 J.R.Hwu, J.J.Huang, F.Y.Tsai, S.C.Tsay, M.H.Hsu, K.C.Hwang, J.C.Horng, J.A.Ho, and C.C.Lin (2009).
Photochemical activities of N-nitroso carboxamides and sulfoximides and their application to DNA cleavage.
  Chemistry, 15, 8742-8750.  
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