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Hydrolase PDB id
1dq3
Jmol
Contents
Protein chain
454 a.a. *
Metals
_ZN
Waters ×332
* Residue conservation analysis
PDB id:
1dq3
Name: Hydrolase
Title: Crystal structure of an archaeal intein-encoded homing endonuclease pi-pfui
Structure: Endonuclease. Chain: a. Engineered: yes
Source: Pyrococcus furiosus. Organism_taxid: 2261. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Resolution:
2.10Å     R-factor:   0.192     R-free:   0.232
Authors: K.Ichiyanagi,Y.Ishino,K.Morikawa
Key ref:
K.Ichiyanagi et al. (2000). Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI. J Mol Biol, 300, 889-901. PubMed id: 10891276 DOI: 10.1006/jmbi.2000.3873
Date:
30-Dec-99     Release date:   05-Jul-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P95484  (P95484_9EURY) -  Ribonucleoside-diphosphate reductase
Seq:
Struc: 		 
Seq:
Struc: 		 
Seq:
Struc: 		 
Seq:
Struc: 		1740 a.a.
454 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.1.17.4.1  - Ribonucleoside-diphosphate reductase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: 2'-deoxyribonucleoside diphosphate + thioredoxin disulfide + H2O = ribonucleoside diphosphate + thioredoxin
2'-deoxyribonucleoside diphosphate
 + thioredoxin disulfide
 + H(2)O
 = ribonucleoside diphosphate
 + thioredoxin
      Cofactor: Iron


Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     intein-mediated protein splicing   1 term 
  Biochemical function     endonuclease activity     1 term  

 

 
    reference    
 
 
DOI no: 10.1006/jmbi.2000.3873 J Mol Biol 300:889-901 (2000)
PubMed id: 10891276  
 
 
Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI.
K.Ichiyanagi, Y.Ishino, M.Ariyoshi, K.Komori, K.Morikawa.
 
  ABSTRACT  
 
Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites.
 
  Selected figure(s)  
 
Figure 5.
Figure 5. (a) Electrostatic potential distribution on the surface of PI-PfuI. The surfaces with negative, neutral, and positive potentials are colored by red, white, and blue, respectively. The four b-hairpins in the endonu- clease domain are indicated with yellow letters. (b) Docking model for the protein-DNA complex. DNA is shown as a ribbon drawing. 		Figure 6.
Figure 6. Possible mechanism of the double-strand break by the two catalytic centers. The DNA backbones of the top (purple) and bottom (red) strands and the protein side-chains of Asp149, Asp173, Glu250, and Lys322 are shown. The arrows indicate the attacks on the scissile phosphate groups. The protein surface is shown in light blue.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 300, 889-901) copyright 2000.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21539790 P.T.Shemella, N.I.Topilina, I.Soga, B.Pereira, G.Belfort, M.Belfort, and S.K.Nayak (2011).
Electronic structure of neighboring extein residue modulates intein C-terminal cleavage activity.
  Biophys J, 100, 2217-2225.  
  19937653 P.Singh, P.Tripathi, and K.Muniyappa (2010).
Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not.
  Protein Sci, 19, 111-123.  
20939100 S.Kalkhof, S.Haehn, M.Paulsson, N.Smyth, J.Meiler, and A.Sinz (2010).
Computational modeling of laminin N-terminal domains using sparse distance constraints from disulfide bonds and chemical cross-linking.
  Proteins, 78, 3409-3427.  
19264795 B.Dassa, N.London, B.L.Stoddard, O.Schueler-Furman, and S.Pietrokovski (2009).
Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family.
  Nucleic Acids Res, 37, 2560-2573.  
19708049 H.D.Mootz (2009).
Split inteins as versatile tools for protein semisynthesis.
  Chembiochem, 10, 2579-2589.  
19153140 H.Li, S.Pellenz, U.Ulge, B.L.Stoddard, and R.J.Monnat (2009).
Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins.
  Nucleic Acids Res, 37, 1650-1662.
PDB code: 3fd2
19136555 J.H.Appleby, K.Zhou, G.Volkmann, and X.Q.Liu (2009).
Novel Split Intein for trans-Splicing Synthetic Peptide onto C Terminus of Protein.
  J Biol Chem, 284, 6194-6199.  
19605345 P.Singh, P.Tripathi, G.H.Silva, A.Pingoud, and K.Muniyappa (2009).
Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA binding, helical distortion, and cleavage compared with a canonical LAGLIDADG homing endonuclease.
  J Biol Chem, 284, 25912-25928.  
18276641 E.Fajardo-Sanchez, F.Stricher, F.Pâques, M.Isalan, and L.Serrano (2008).
Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequences.
  Nucleic Acids Res, 36, 2163-2173.  
17999959 J.Prieto, J.C.Epinat, P.Redondo, E.Ramos, D.Padró, F.Cédrone, G.Montoya, F.Pâques, and F.J.Blanco (2008).
Generation and analysis of mesophilic variants of the thermostable archaeal I-DmoI homing endonuclease.
  J Biol Chem, 283, 4364-4374.  
17452357 J.Prieto, P.Redondo, D.Padró, S.Arnould, J.C.Epinat, F.Pâques, F.J.Blanco, and G.Montoya (2007).
The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage.
  Nucleic Acids Res, 35, 3262-3271.
PDB code: 2o7m
17586768 M.A.Johnson, M.W.Southworth, T.Herrmann, L.Brace, F.B.Perler, and K.Wüthrich (2007).
NMR structure of a KlbA intein precursor from Methanococcus jannaschii.
  Protein Sci, 16, 1316-1328.
PDB codes: 2jmz 2jnq
  18084082 P.Redondo, J.Prieto, E.Ramos, F.J.Blanco, and G.Montoya (2007).
Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 63, 1017-1020.  
17085503 P.Shemella, B.Pereira, Y.Zhang, P.Van Roey, G.Belfort, S.Garde, and S.K.Nayak (2007).
Mechanism for intein C-terminal cleavage: a proposal from quantum mechanical calculations.
  Biophys J, 92, 847-853.  
17254599 P.Van Roey, B.Pereira, Z.Li, K.Hiraga, M.Belfort, and V.Derbyshire (2007).
Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue.
  J Mol Biol, 367, 162-173.
PDB codes: 2imz 2in0 2in8 2in9
17639349 Z.Xie, W.Li, Y.Tian, G.Liu, and H.Tan (2007).
Identification and characterization of sawC, a whiA-like gene, essential for sporulation in Streptomyces ansochromogenes.
  Arch Microbiol, 188, 575-582.  
16493661 H.Matsumura, H.Takahashi, T.Inoue, T.Yamamoto, H.Hashimoto, M.Nishioka, S.Fujiwara, M.Takagi, T.Imanaka, and Y.Kai (2006).
Crystal structure of intein homing endonuclease II encoded in DNA polymerase gene from hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1.
  Proteins, 63, 711-715.
PDB codes: 2cw7 2cw8
16830226 J.Yang, T.V.Henry-Smith, and M.Qi (2006).
Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment.
  Transgenic Res, 15, 583-593.  
16895922 K.S.Makarova, N.V.Grishin, and E.V.Koonin (2006).
The HicAB cassette, a putative novel, RNA-targeting toxin-antitoxin system in archaea and bacteria.
  Bioinformatics, 22, 2581-2584.  
15020462 A.Bakhrat, M.S.Jurica, B.L.Stoddard, and D.Raveh (2004).
Homology modeling and mutational analysis of Ho endonuclease of yeast.
  Genetics, 166, 721-728.  
15087498 A.Romanelli, A.Shekhtman, D.Cowburn, and T.W.Muir (2004).
Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction.
  Proc Natl Acad Sci U S A, 101, 6397-6402.  
15190132 G.H.Silva, and M.Belfort (2004).
Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI.
  Nucleic Acids Res, 32, 3156-3168.  
15189989 J.D.Wuitschick, P.R.Lindstrom, A.E.Meyer, and K.M.Karrer (2004).
Homing endonucleases encoded by germ line-limited genes in Tetrahymena thermophila have APETELA2 DNA binding domains.
  Eukaryot Cell, 3, 685-694.  
15099733 R.Aroul-Selvam, T.Hubbard, and R.Sasidharan (2004).
Domain insertions in protein structures.
  J Mol Biol, 338, 633-641.  
14760742 S.Steuer, V.Pingoud, A.Pingoud, and W.Wende (2004).
Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity.
  Chembiochem, 5, 206-213.  
15502310 T.Imagawa, H.Nakayama, N.Katunuma, H.Sakuraba, T.Ohshima, T.Itoh, Y.Sako, N.Nomura, and H.Tsuge (2004).
Crystallization and preliminary X-ray diffraction analysis of homing endonuclease I-Tsp061I.
  Acta Crystallogr D Biol Crystallogr, 60, 2006-2008.  
15194682 W.Sun, J.Yang, and X.Q.Liu (2004).
Synthetic two-piece and three-piece split inteins for protein trans-splicing.
  J Biol Chem, 279, 35281-35286.  
12771221 J.C.Epinat, S.Arnould, P.Chames, P.Rochaix, D.Desfontaines, C.Puzin, A.Patin, A.Zanghellini, F.Pâques, and E.Lacroix (2003).
A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.
  Nucleic Acids Res, 31, 2952-2962.  
14633971 J.M.Bolduc, P.C.Spiegel, P.Chatterjee, K.L.Brady, M.E.Downing, M.G.Caprara, R.B.Waring, and B.L.Stoddard (2003).
Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.
  Genes Dev, 17, 2875-2888.
PDB code: 1p8k
12654010 W.J.Geese, Y.K.Kwon, X.Wen, and R.B.Waring (2003).
In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.
  Eur J Biochem, 270, 1543-1554.  
12975359 X.Q.Liu, J.Yang, and Q.Meng (2003).
Four inteins and three group II introns encoded in a bacterial ribonucleotide reductase gene.
  J Biol Chem, 278, 46826-46831.  
12878593 Y.Ding, M.Q.Xu, I.Ghosh, X.Chen, S.Ferrandon, G.Lesage, and Z.Rao (2003).
Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing.
  J Biol Chem, 278, 39133-39142.
PDB code: 1mi8
12419232 B.S.Chevalier, T.Kortemme, M.S.Chadsey, D.Baker, R.J.Monnat, and B.L.Stoddard (2002).
Design, activity, and structure of a highly specific artificial endonuclease.
  Mol Cell, 10, 895-905.
PDB code: 1mow
12219083 C.M.Moure, F.S.Gimble, and F.A.Quiocho (2002).
Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence.
  Nat Struct Biol, 9, 764-770.
PDB codes: 1lws 1lwt
12235380 E.Werner, W.Wende, A.Pingoud, and U.Heinemann (2002).
High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI.
  Nucleic Acids Res, 30, 3962-3971.
PDB code: 1gpp
12202772 L.M.Seligman, K.M.Chisholm, B.S.Chevalier, M.S.Chadsey, S.T.Edwards, J.H.Savage, and A.L.Veillet (2002).
Mutations altering the cleavage specificity of a homing endonuclease.
  Nucleic Acids Res, 30, 3870-3879.  
11850426 N.Guhan, and K.Muniyappa (2002).
Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity.
  J Biol Chem, 277, 16257-16264.  
12077443 X.Chen, M.Q.Xu, Y.Ding, S.Ferrandon, and Z.Rao (2002).
Purification and initial crystallization studies of a DnaB intein from Synechocystis sp. PCC 6803.
  Acta Crystallogr D Biol Crystallogr, 58, 1201-1203.  
12077444 X.Chen, M.Q.Xu, Y.Ding, S.Ferrandon, and Z.Rao (2002).
Crystallographic study of a naturally occurring trans-splicing intein from Synechocystis sp. PCC 6803.
  Acta Crystallogr D Biol Crystallogr, 58, 1204-1206.  
11557808 B.S.Chevalier, and B.L.Stoddard (2001).
Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
  Nucleic Acids Res, 29, 3757-3774.  
11600710 F.S.Gimble (2001).
Degeneration of a homing endonuclease and its target sequence in a wild yeast strain.
  Nucleic Acids Res, 29, 4215-4223.  
11160929 P.Lucas, C.Otis, J.P.Mercier, M.Turmel, and C.Lemieux (2001).
Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases.
  Nucleic Acids Res, 29, 960-969.  
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.  
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.