PDBsum entry 1m0d

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Hydrolase PDB id
Protein chains
129 a.a. *
SO4 ×4
_MN ×8
Waters ×473
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of bacteriophage t7 endonuclease i with a wild-type active site and bound manganese ions
Structure: Endodeoxyribonuclease i. Chain: a, b, c, d. Fragment: residues 12-149. Synonym: endonuclease. Engineered: yes
Source: Enterobacteria phage t7. Organism_taxid: 10760. Gene: endonuclease i. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Octamer (from PQS)
1.90Å     R-factor:   0.210     R-free:   0.234
Authors: J.M.Hadden,A.C.Declais,S.E.Phillips,D.M.Lilley
Key ref:
J.M.Hadden et al. (2002). Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I. EMBO J, 21, 3505-3515. PubMed id: 12093751 DOI: 10.1093/emboj/cdf337
12-Jun-02     Release date:   10-Jul-02    
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Protein chains
Pfam   ArchSchema ?
P00641  (ENRN_BPT7) -  Endonuclease I
149 a.a.
129 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Deoxyribonuclease Iv.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endonucleolytic cleavage to 5'-phosphooligonucleotide end-products.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     viral reproduction   6 terms 
  Biochemical function     hydrolase activity     6 terms  


DOI no: 10.1093/emboj/cdf337 EMBO J 21:3505-3515 (2002)
PubMed id: 12093751  
Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I.
J.M.Hadden, A.C.Déclais, S.E.Phillips, D.M.Lilley.
T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.
  Selected figure(s)  
Figure 2.
Figure 2 Stereo view showing a ribbon representation of the structure of T7 endonuclease I with bound manganese ions. One subunit is drawn in light blue and the other in green. Active site residues are depicted in red (acidic amino acids) and blue (lysine). Manganese ions are represented as yellow spheres. All figures depicting the crystal structure of endonuclease I were generated using the program SPOCK (Christopher, 1998).
Figure 3.
Figure 3 Stereo images showing close-up views of the active site of T7 endonuclease I with bound manganese and sulfate ions. Endonuclease I subunits are shown in light blue and green. Active site residues are depicted in red (acidic residues and Thr66 CO) and blue (lysine). Manganese and sulfate ions are represented in yellow. (A) F[o] - F[c] electron density map in the vicinity of the active site contoured at 5.5 with the manganese and sulfate ions and active site water molecules omitted from the model. Difference density can clearly be seen for the two manganese ions, the sulfate moiety and the three water molecules that are strongly associated with the metal bound at site 1. Reduction of the level to 3.0 would reveal electron density for the additional water molecules associated with metal site 2 (not shown). (B) The metal-ligating residues, manganese ions, sulfate ion and local solvent structure. Coordinating water molecules are shown in light blue. Potential interactions are highlighted with dashed lines.
  The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 3505-3515) copyright 2002.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19161971 F.Xie, and C.M.Dupureur (2009).
Kinetic analysis of product release and metal ions in a metallonuclease.
  Arch Biochem Biophys, 483, 1-9.  
18160275 A.C.Déclais, and D.M.Lilley (2008).
New insight into the recognition of branched DNA structure by junction-resolving enzymes.
  Curr Opin Struct Biol, 18, 86-95.  
18552127 Z.Guo, and D.Eisenberg (2008).
The structure of a fibril-forming sequence, NNQQNY, in the context of a globular fold.
  Protein Sci, 17, 1617-1623.
PDB code: 3cae
17308914 G.A.Papadakos, H.Nastri, P.Riggs, and C.M.Dupureur (2007).
Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.
  J Biol Inorg Chem, 12, 557-569.  
17873858 J.M.Hadden, A.C.Déclais, S.B.Carr, D.M.Lilley, and S.E.Phillips (2007).
The structural basis of Holliday junction resolution by T7 endonuclease I.
  Nature, 449, 621-624.
PDB code: 2pfj
17214552 L.Mones, I.Simon, and M.Fuxreiter (2007).
Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism.
  Biol Chem, 388, 73-78.  
17584608 P.M.Gordon, R.Fong, and J.A.Piccirilli (2007).
A second divalent metal ion in the group II intron reaction center.
  Chem Biol, 14, 607-612.  
16732569 A.M.Polyanichko, E.V.Chikhirzhina, V.V.Andrushchenko, V.I.Vorob'ev, and H.Wieser (2006).
The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies.
  Biopolymers, 83, 182-192.  
15972856 L.N.Kinch, K.Ginalski, L.Rychlewski, and N.V.Grishin (2005).
Identification of novel restriction endonuclease-like fold families among hypothetical proteins.
  Nucleic Acids Res, 33, 3598-3605.  
14872058 A.M.Polyanichko, V.V.Andrushchenko, E.V.Chikhirzhina, V.I.Vorob'ev, and H.Wieser (2004).
The effect of manganese(II) on DNA structure: electronic and vibrational circular dichroism studies.
  Nucleic Acids Res, 32, 989-996.  
15479781 C.L.Middleton, J.L.Parker, D.J.Richard, M.F.White, and C.S.Bond (2004).
Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje.
  Nucleic Acids Res, 32, 5442-5451.
PDB codes: 1ob8 1ob9
15613597 J.Yu, T.Ha, and K.Schulten (2004).
Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.
  Nucleic Acids Res, 32, 6683-6695.  
12628932 A.C.Déclais, J.M.Fogg, A.D.Freeman, F.Coste, J.M.Hadden, S.E.Phillips, and D.M.Lilley (2003).
The complex between a four-way DNA junction and T7 endonuclease I.
  EMBO J, 22, 1398-1409.  
14732929 M.J.Cliff, and J.E.Ladbury (2003).
A survey of the year 2002 literature on applications of isothermal titration calorimetry.
  J Mol Recognit, 16, 383-391.  
12606565 M.R.Tock, E.Frary, J.R.Sayers, and J.A.Grasby (2003).
Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.
  EMBO J, 22, 995.  
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.