PDBsum entry 1b94

Go to PDB code: 
protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
Protein chains
244 a.a.
_CA ×2
Waters ×350
PDB id:
Name: Hydrolase/DNA
Title: Restriction endonuclease ecorv with calcium
Structure: DNA (5'-d( Ap Ap Ap Gp Ap Tp Ap Tp Cp Tp T)-3'). Chain: c, d. Engineered: yes. Restriction endonuclease ecorv. Chain: a, b. Engineered: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Cellular_location: cytoplasm. Gene: ecorvr. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
1.90Å     R-factor:   0.206     R-free:   0.270
Authors: M.P.Thomas,S.E.Halford,R.L.Brady
Key ref: M.P.Thomas et al. (1999). Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group. Nucleic Acids Res, 27, 3438-3445. PubMed id: 10446231
19-Feb-99     Release date:   26-Feb-99    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
245 a.a.
244 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!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     hydrolase activity     6 terms  


Nucleic Acids Res 27:3438-3445 (1999)
PubMed id: 10446231  
Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.
M.P.Thomas, R.L.Brady, S.E.Halford, R.B.Sessions, G.S.Baldwin.
Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

Literature references that cite this PDB file's key reference

  PubMed id Reference
20047562 S.Kitamura, K.Fujishima, A.Sato, D.Tsuchiya, M.Tomita, and A.Kanai (2010).
Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus.
  Biochem J, 426, 337-344.  
19389725 J.Ashworth, and D.Baker (2009).
Assessment of the optimization of affinity and specificity at protein-DNA interfaces.
  Nucleic Acids Res, 37, e73.  
18436236 A.K.Mantha, N.Oezguen, K.K.Bhakat, T.Izumi, W.Braun, and S.Mitra (2008).
Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1.
  J Mol Biol, 379, 28-37.  
17427952 N.Oezguen, C.H.Schein, S.R.Peddi, T.D.Power, T.Izumi, and W.Braun (2007).
A "moving metal mechanism" for substrate cleavage by the DNA repair endonuclease APE-1.
  Proteins, 68, 313-323.  
16710870 K.D.Roberts, J.N.Lambert, N.J.Ede, and A.M.Bray (2006).
Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.
  J Pept Sci, 12, 525-532.  
16154091 N.McGregor, S.Ayora, S.Sedelnikova, B.Carrasco, J.C.Alonso, P.Thaw, and J.Rafferty (2005).
The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage.
  Structure, 13, 1341-1351.
PDB code: 1zp7
14725771 G.Paillard, and R.Lavery (2004).
Analyzing protein-DNA recognition mechanisms.
  Structure, 12, 113-122.  
11557805 A.Pingoud, and A.Jeltsch (2001).
Structure and function of type II restriction endonucleases.
  Nucleic Acids Res, 29, 3705-3727.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.