PDBsum entry 1suz

Go to PDB code: 
protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
Jmol PyMol
Protein chains
243 a.a.
_NA ×2
_MG ×2
Waters ×303
PDB id:
Name: Hydrolase/DNA
Title: The structure of k92a ecorv bound to cognate DNA and mg2+
Structure: 5'-d( C Ap Ap Gp Ap Tp Ap Tp Cp Tp T)-3'. Chain: c, d. Engineered: yes. Type ii restriction enzyme ecorv. Chain: a, b. Synonym: endonuclease ecorv, r.Ecorv. Engineered: yes. Mutation: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Gene: ecorvr. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
1.80Å     R-factor:   0.197     R-free:   0.244
Authors: N.C.Horton,J.J.Perona
Key ref:
N.C.Horton and J.J.Perona (2004). DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites. Biochemistry, 43, 6841-6857. PubMed id: 15170321 DOI: 10.1021/bi0499056
26-Mar-04     Release date:   06-Apr-04    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
245 a.a.
243 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   2 terms 
  Biochemical function     hydrolase activity     5 terms  


DOI no: 10.1021/bi0499056 Biochemistry 43:6841-6857 (2004)
PubMed id: 15170321  
DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites.
N.C.Horton, J.J.Perona.
Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.

Literature references that cite this PDB file's key reference

  PubMed id Reference
20854710 W.Yang (2011).
Nucleases: diversity of structure, function and mechanism.
  Q Rev Biophys, 44, 1.  
20592097 T.Crépin, A.Dias, A.Palencia, C.Swale, S.Cusack, and R.W.Ruigrok (2010).
Mutational and metal binding analysis of the endonuclease domain of the influenza virus polymerase PA subunit.
  J Virol, 84, 9096-9104.  
19194459 A.Dias, D.Bouvier, T.Crépin, A.A.McCarthy, D.J.Hart, F.Baudin, S.Cusack, and R.W.Ruigrok (2009).
The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.
  Nature, 458, 914-918.
PDB code: 2w69
19089001 C.Liu, and L.Wang (2009).
DNA hydrolytic cleavage catalyzed by synthetic multinuclear metallonucleases.
  Dalton Trans, (), 227-239.  
19188255 C.Sissi, and M.Palumbo (2009).
Effects of magnesium and related divalent metal ions in topoisomerase structure and function.
  Nucleic Acids Res, 37, 702-711.  
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.  
18762194 A.C.Babic, E.J.Little, V.M.Manohar, J.Bitinaite, and N.C.Horton (2008).
DNA distortion and specificity in a sequence-specific endonuclease.
  J Mol Biol, 383, 186-204.
PDB codes: 3e3y 3e40 3e41 3e42 3e43 3e44 3e45
18261473 C.M.Dupureur (2008).
Roles of metal ions in nucleases.
  Curr Opin Chem Biol, 12, 250-255.  
18424798 C.M.Moure, F.S.Gimble, and F.A.Quiocho (2008).
Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway.
  Nucleic Acids Res, 36, 3287-3296.
PDB codes: 3c0w 3c0x
18975919 F.Xie, S.H.Qureshi, G.A.Papadakos, and C.M.Dupureur (2008).
One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism.
  Biochemistry, 47, 12540-12550.  
18697748 K.Syson, C.Tomlinson, B.R.Chapados, J.R.Sayers, J.A.Tainer, N.H.Williams, and J.A.Grasby (2008).
Three metal ions participate in the reaction catalyzed by t5 flap endonuclease.
  J Biol Chem, 283, 28741-28746.  
18701646 P.W.Dunten, E.J.Little, M.T.Gregory, V.M.Manohar, M.Dalton, D.Hough, J.Bitinaite, and N.C.Horton (2008).
The structure of SgrAI bound to DNA; recognition of an 8 base pair target.
  Nucleic Acids Res, 36, 5405-5416.
PDB codes: 3dpg 3dvo 3dw9
18953336 W.Yang (2008).
An equivalent metal ion in one- and two-metal-ion catalysis.
  Nat Struct Mol Biol, 15, 1228-1231.  
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.  
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.  
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.  
16981705 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.
PDB code: 2ge5
16601679 M.Nowotny, and W.Yang (2006).
Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release.
  EMBO J, 25, 1924-1933.
PDB codes: 2g8f 2g8h 2g8i 2g8k 2g8u 2g8v 2g8w
16600865 W.Yang, J.Y.Lee, and M.Nowotny (2006).
Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.
  Mol Cell, 22, 5.  
16372100 Y.M.Chung, B.Raman, D.S.Kim, and K.H.Ahn (2006).
Fluorescence modulation in anion sensing by introducing intramolecular H-bonding interactions in host-guest adducts.
  Chem Commun (Camb), (), 186-188.  
16209953 J.Y.Lee, J.Chang, N.Joseph, R.Ghirlando, D.N.Rao, and W.Yang (2005).
MutH complexed with hemi- and unmethylated DNAs: coupling base recognition and DNA cleavage.
  Mol Cell, 20, 155-166.
PDB codes: 2aoq 2aor
15741179 K.Range, E.Mayaan, L.J.Maher, and D.M.York (2005).
The contribution of phosphate-phosphate repulsions to the free energy of DNA bending.
  Nucleic Acids Res, 33, 1257-1268.  
15699182 W.Sun, A.Pertzev, and A.W.Nicholson (2005).
Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis.
  Nucleic Acids Res, 33, 807-815.  
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.