PDBsum entry 3pvi

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protein dna_rna Protein-protein interface(s) links
Hydrolase/DNA PDB id
Protein chains
156 a.a.
Waters ×269
PDB id:
Name: Hydrolase/DNA
Title: D34g mutant of pvuii endonuclease complexed with cognate DNA shows that asp34 is directly involved in DNA recognition and indirectly involved in catalysis
Structure: DNA (5'- d( Tp Gp Ap Cp Cp Ap Gp Cp Tp Gp Gp Tp C)-3'). Chain: c, d. Synonym: cognate oligonucleotide. Engineered: yes. Protein (pvuii endonuclease). Chain: a, b. Engineered: yes. Mutation: yes
Source: Synthetic: yes. Proteus vulgaris. Organism_taxid: 585. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
1.59Å     R-factor:   0.205     R-free:   0.253
Authors: J.R.Horton,X.Cheng
Key ref:
J.R.Horton et al. (1998). Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis. J Mol Biol, 284, 1491-1504. PubMed id: 9878366 DOI: 10.1006/jmbi.1998.2269
09-Oct-98     Release date:   14-Oct-98    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P23657  (T2P2_PROHU) -  Type-2 restriction enzyme PvuII
157 a.a.
156 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+)


DOI no: 10.1006/jmbi.1998.2269 J Mol Biol 284:1491-1504 (1998)
PubMed id: 9878366  
Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.
J.R.Horton, H.G.Nastri, P.D.Riggs, X.Cheng.
The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.
  Selected figure(s)  
Figure 1.
Figure 1. D34G homodimer complexed with DNA. The subunits are colored red, yellow, and green to illus- trate the dimerization, catalytic, and DNA recognition regions, respectively. The DNA backbone is colored orange. Ball and stick representations of some amino acids are included: Asp58 in yellow, His85 in red, and Tyr94 in green. The Gly34 C a atoms are shown as green dotted spheres. The model was drawn using the Ribbons (Carson, 1997) program.
Figure 5.
Figure 5. Asymmetric active sites of the homodimeric D34G/DNA. (a) A portion of the 2Fo - Fc electron density (light blue) at 1.5s level showing the ``primed'' active site. The water molecules are labeled a,b,c,d in white lettering. Amino acid and nucleic acid residues, are labeled with black lettering. The scissile bond is indicated by an arrow. (b) A portion of the 2Fo - Fc electron density (light blue) at 1.5s level showing the ``unprimed'' active site. The lettering is according to Figure 5(a).
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 284, 1491-1504) copyright 1998.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19289051 S.Balasubramanian, F.Xu, and W.K.Olson (2009).
DNA sequence-directed organization of chromatin: structure-based computational analysis of nucleosome-binding sequences.
  Biophys J, 96, 2245-2260.  
17557334 S.J.Kelly, J.Li, P.Setlow, and M.J.Jedrzejas (2007).
Structure, flexibility, and mechanism of the Bacillus stearothermophilus RecU Holliday junction resolvase.
  Proteins, 68, 961-971.
PDB code: 2fco
16839194 J.Gu, M.Gribskov, and P.E.Bourne (2006).
Wiggle-predicting functionally flexible regions from primary sequence.
  PLoS Comput Biol, 2, e90.  
15684412 S.D.Pawlak, M.Radlinska, A.A.Chmiel, J.M.Bujnicki, and K.J.Skowronek (2005).
Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.
  Nucleic Acids Res, 33, 661-671.  
14580211 L.M.Bowen, and C.M.Dupureur (2003).
Investigation of restriction enzyme cofactor requirements: a relationship between metal ion properties and sequence specificity.
  Biochemistry, 42, 12643-12653.  
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.  
11842098 S.Grazulis, M.Deibert, R.Rimseliene, R.Skirgaila, G.Sasnauskas, A.Lagunavicius, V.Repin, C.Urbanke, R.Huber, and V.Siksnys (2002).
Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence.
  Nucleic Acids Res, 30, 876-885.
PDB code: 1knv
11557805 A.Pingoud, and A.Jeltsch (2001).
Structure and function of type II restriction endonucleases.
  Nucleic Acids Res, 29, 3705-3727.  
10931930 M.R.Rice, and R.M.Blumenthal (2000).
Recognition of native DNA methylation by the PvuII restriction endonuclease.
  Nucleic Acids Res, 28, 3143-3150.  
10593932 D.T.Bilcock, L.E.Daniels, A.J.Bath, and S.E.Halford (1999).
Reactions of type II restriction endonucleases with 8-base pair recognition sites.
  J Biol Chem, 274, 36379-36386.  
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