PDBsum entry 1bss

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protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
Protein chains
230 a.a.
_CA ×2
Waters ×166
PDB id:
Name: Hydrolase/DNA
Title: Ecorv-t93a/DNA/ca2+
Structure: 5'-d( Ap Ap Ap Gp Ap Tp Ap Tp Cp Tp T)-3'. Chain: c, d. Engineered: yes. Ecorv endonuclease. Chain: a, b. Engineered: yes. Mutation: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Tetramer (from PQS)
2.15Å     R-factor:   0.184     R-free:   0.246
Authors: J.J.Perona,N.C.Horton
Key ref:
N.C.Horton et al. (1998). Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. Proc Natl Acad Sci U S A, 95, 13489-13494. PubMed id: 9811827 DOI: 10.1073/pnas.95.23.13489
30-Aug-98     Release date:   02-Sep-98    
Go to PROCHECK summary

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

 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  


DOI no: 10.1073/pnas.95.23.13489 Proc Natl Acad Sci U S A 95:13489-13494 (1998)
PubMed id: 9811827  
Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
N.C.Horton, K.J.Newberry, J.J.Perona.
The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
  Selected figure(s)  
Figure 1.
Fig. 1. Superposition of the structures of wild-type EcoRV (red) and the T93A mutant (white, green) based on sugar-phosphate atoms of the DNA at the center base pairs of the target site. Thr-93 in the wild-type enzyme is shown in magenta, and the active-site side chains are indicated. The blue spheres indicate the Ca^2+ binding sites observed in this mutant structure; the yellow sphere shows the position of the Ca^2+ binding site previously found in the wild-type structure.
Figure 3.
Fig. 3. Detailed structure of the Ca^2+ binding sites in subunit I of the EcoRV T93A-DNA-Ca^2+ complex. Metal-ligand distances and distances between the electronegative atoms for selected hydrogen bonds are indicated in Å.
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20861000 M.Firczuk, M.Wojciechowski, H.Czapinska, and M.Bochtler (2011).
DNA intercalation without flipping in the specific ThaI-DNA complex.
  Nucleic Acids Res, 39, 744-754.
PDB code: 3ndh
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.  
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
18843295 V.Pena, A.Rozov, P.Fabrizio, R.Lührmann, and M.C.Wahl (2008).
Structure and function of an RNase H domain at the heart of the spliceosome.
  EMBO J, 27, 2929-2940.
PDB codes: 3e9l 3e9o 3e9p
18268334 Z.Du, J.K.Lee, R.Tjhen, R.M.Stroud, and T.L.James (2008).
Structural and biochemical insights into the dicing mechanism of mouse Dicer: a conserved lysine is critical for dsRNA cleavage.
  Proc Natl Acad Sci U S A, 105, 2391-2396.
PDB codes: 3c4b 3c4t
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.  
16195548 Q.S.Xu, R.J.Roberts, and H.C.Guo (2005).
Two crystal forms of the restriction enzyme MspI-DNA complex show the same novel structure.
  Protein Sci, 14, 2590-2600.
PDB code: 1yfi
15341737 Q.S.Xu, R.B.Kucera, R.J.Roberts, and H.C.Guo (2004).
An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site.
  Structure, 12, 1741-1747.
PDB code: 1sa3
12732532 I.Mruk, and T.Kaczorowski (2003).
Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs.
  Appl Environ Microbiol, 69, 2638-2650.  
12142452 M.Fuxreiter, and I.Simon (2002).
Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases.
  Protein Sci, 11, 1978-1983.  
11557805 A.Pingoud, and A.Jeltsch (2001).
Structure and function of type II restriction endonucleases.
  Nucleic Acids Res, 29, 3705-3727.  
11179886 C.M.Lukacs, and A.K.Aggarwal (2001).
BglII and MunI: what a difference a base makes.
  Curr Opin Struct Biol, 11, 14-18.  
11250198 M.J.van der Woerd, J.J.Pelletier, S.Xu, and A.M.Friedman (2001).
Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis.
  Structure, 9, 133-144.
PDB code: 1dc1
11557809 S.E.Tsutakawa, and K.Morikawa (2001).
The structural basis of damaged DNA recognition and endonucleolytic cleavage for very short patch repair endonuclease.
  Nucleic Acids Res, 29, 3775-3783.  
11327870 S.L.Reid, D.Parry, H.H.Liu, and B.A.Connolly (2001).
Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization.
  Biochemistry, 40, 2484-2494.  
11257528 W.Cao, and J.Lu (2001).
Exploring the catalytic center of TaqI endonuclease: rescuing catalytic activity by double mutations and Mn2+.
  Biochim Biophys Acta, 1546, 253-260.  
10801972 N.C.Horton, and J.J.Perona (2000).
Crystallographic snapshots along a protein-induced DNA-bending pathway.
  Proc Natl Acad Sci U S A, 97, 5729-5734.
PDB codes: 1eoo 1eop
10856254 Q.Huai, J.D.Colandene, Y.Chen, F.Luo, Y.Zhao, M.D.Topal, and H.Ke (2000).
Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase.
  EMBO J, 19, 3110-3118.
PDB code: 1ev7
10864040 S.O.Shan, and D.Herschlag (2000).
An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction.
  RNA, 6, 795-813.  
11123916 S.Schöttler, W.Wende, V.Pingoud, and A.Pingoud (2000).
Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI.
  Biochemistry, 39, 15895-15900.  
  10739241 W.Dall'Acqua, and P.Carter (2000).
Substrate-assisted catalysis: molecular basis and biological significance.
  Protein Sci, 9, 1-9.  
10387089 A.M.Martin, N.C.Horton, S.Lusetti, N.O.Reich, and J.J.Perona (1999).
Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.
  Biochemistry, 38, 8430-8439.  
10350476 M.D.Sam, and J.J.Perona (1999).
Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.
  Biochemistry, 38, 6576-6586.  
10508668 R.A.Kovall, and B.W.Matthews (1999).
Type II restriction endonucleases: structural, functional and evolutionary relationships.
  Curr Opin Chem Biol, 3, 578-583.  
10360178 S.E.Tsutakawa, T.Muto, T.Kawate, H.Jingami, N.Kunishima, M.Ariyoshi, D.Kohda, M.Nakagawa, and K.Morikawa (1999).
Crystallographic and functional studies of very short patch repair endonuclease.
  Mol Cell, 3, 621-628.
PDB code: 1vsr
10587440 S.J.Mannino, C.L.Jenkins, and R.T.Raines (1999).
Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI.
  Biochemistry, 38, 16178-16186.  
10535916 S.Shan, A.Yoshida, S.Sun, J.A.Piccirilli, and D.Herschlag (1999).
Three metal ions at the active site of the Tetrahymena group I ribozyme.
  Proc Natl Acad Sci U S A, 96, 12299-12304.  
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.