Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site

BackgroundCatalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.ResultsR.Hpy188I is a Type II REase with unknown structure. PSI-BLAST searches of the non-redundant protein sequence database reveal only 1 homolog (R.HpyF17I, with nearly identical amino acid sequence and the same DNA sequence specificity). Standard application of state-of-the-art protein fold-recognition methods failed to predict the relationship of R.Hpy188I to proteins with known structure or to other protein families. In order to increase the amount of evolutionary information in the multiple sequence alignment, we have expanded our sequence database searches to include sequences from metagenomics projects. This search resulted in identification of 23 further members of R.Hpy188I family, both from metagenomics and the non-redundant database. Moreover, fold-recognition analysis of the extended R.Hpy188I family revealed its relationship to the GIY-YIG domain and allowed for computational modeling of the R.Hpy188I structure. Analysis of the R.Hpy188I model in the light of sequence conservation among its homologs revealed an unusual variant of the active site, in which the typical Tyr residue of the YIG half-motif had been substituted by a Lys residue. Moreover, some of its homologs have the otherwise invariant Arg residue in a non-homologous position in sequence that nonetheless allows for spatial conservation of the guanidino group potentially involved in phosphate binding.ConclusionThe present study eliminates a significant "white spot" on the structural map of REases. It also provides important insight into sequence-structure-function relationships in the GIY-YIG nuclease superfamily. Our results reveal that in the case of proteins with no or few detectable homologs in the standard "non-redundant" database, it is useful to expand this database by adding the metagenomic sequences, which may provide evolutionary linkage to detect more remote homologs.

[1]  Marcin Feder,et al.  Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site , 2005, BMC Genomics.

[2]  Itay Mayrose,et al.  ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures , 2005, Nucleic Acids Res..

[3]  Janusz M. Bujnicki,et al.  COLORADO3D, a web server for the visual analysis of protein structures , 2004, Nucleic Acids Res..

[4]  Janusz M Bujnicki,et al.  Generalized protein structure prediction based on combination of fold‐recognition with de novo folding and evaluation of models , 2005, Proteins.

[5]  Marcin Feder,et al.  The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function , 2005, BMC Bioinformatics.

[6]  Marcin Feder,et al.  Phylogenomic analysis of the GIY-YIG nuclease superfamily , 2006, BMC Genomics.

[7]  J. Bujnicki,et al.  Polyphyletic evolution of type II restriction enzymes revisited: two independent sources of second-hand folds revealed. , 2001, Trends in biochemical sciences.

[8]  Arne Elofsson,et al.  Pcons5: combining consensus, structural evaluation and fold recognition scores , 2005, Bioinform..

[9]  C Kooperberg,et al.  Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. , 1997, Journal of molecular biology.

[10]  Janusz M Bujnicki,et al.  Crystallographic and bioinformatic studies on restriction endonucleases: inference of evolutionary relationships in the "midnight zone" of homology. , 2003, Current protein & peptide science.

[11]  V. Šikšnys,et al.  Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically , 2007, Nucleic acids research.

[12]  Janusz M Bujnicki,et al.  Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily. , 2004, Nucleic acids research.

[13]  J. Bujnicki,et al.  Specificity Changes in the Evolution of Type II Restriction Endonucleases , 2005, Journal of Biological Chemistry.

[14]  E V Koonin,et al.  SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. , 2000, Nucleic acids research.

[15]  A. Kolinski.  Protein modeling and structure prediction with a reduced representation. , 2004, Acta biochimica Polonica.

[16]  I. Kobayashi,et al.  Evolution of sequence recognition by restriction-modification enzymes: selective pressure for specificity decrease. , 2000, Molecular biology and evolution.

[17]  J. Bujnicki,et al.  Identification of a single HNH active site in type IIS restriction endonuclease Eco31I. , 2007, Journal of molecular biology.

[18]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[19]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[20]  Liam J. McGuffin,et al.  Improvement of the GenTHREADER Method for Genomic Fold Recognition , 2003, Bioinform..

[21]  Leszek Rychlewski,et al.  LiveBench‐8: The large‐scale, continuous assessment of automated protein structure prediction , 2005, Protein science : a publication of the Protein Society.

[22]  D Fischer,et al.  LiveBench‐2: Large‐scale automated evaluation of protein structure prediction servers , 2001, Proteins.

[23]  R J Roberts,et al.  Restriction endonucleases. , 1976, CRC critical reviews in biochemistry.

[24]  Janusz M Bujnicki,et al.  A model of restriction endonuclease MvaI in complex with DNA: A template for interpretation of experimental data and a guide for specificity engineering , 2007, Proteins.

[25]  I Uchiyama,et al.  Insertion with long target duplication: a mechanism for gene mobility suggested from comparison of two related bacterial genomes. , 2000, Gene.

[26]  J. Heitman,et al.  A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. , 2003, Nucleic acids research.

[27]  Marcin Feder,et al.  A “FRankenstein's monster” approach to comparative modeling: Merging the finest fragments of Fold‐Recognition models and iterative model refinement aided by 3D structure evaluation , 2003, Proteins.

[28]  Y. Iwasa,et al.  Genetic Addiction: Selfish Gene's Strategy for Symbiosis in the Genome , 2006, Genetics.

[29]  A. Elofsson,et al.  Can correct protein models be identified? , 2003, Protein science : a publication of the Protein Society.

[30]  Robert Huber,et al.  Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[31]  K. Katoh,et al.  MAFFT version 5: improvement in accuracy of multiple sequence alignment , 2005, Nucleic acids research.

[32]  J. Bujnicki.  Understanding the evolution of restriction-modification systems: clues from sequence and structure comparisons. , 2001, Acta biochimica Polonica.

[33]  Benjamin J. Raphael,et al.  The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families , 2007, PLoS biology.

[34]  H. Viadiu,et al.  A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA , 2005, The EMBO journal.

[35]  V. Šikšnys,et al.  Novel Subtype of Type IIs Restriction Enzymes , 2000, The Journal of Biological Chemistry.

[36]  Andrzej Kolinski,et al.  Protein fragment reconstruction using various modeling techniques , 2003, J. Comput. Aided Mol. Des..

[37]  I. Kobayashi.  Restriction-Modification Systems as Minimal Forms of Life , 2004 .

[38]  M. Blaser,et al.  Identification of type II restriction and modification systems in Helicobacter pylori reveals their substantial diversity among strains. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  K Kusano,et al.  Selfish behavior of restriction-modification systems , 1995, Science.

[40]  Johannes Söding,et al.  The HHpred interactive server for protein homology detection and structure prediction , 2005, Nucleic Acids Res..

[41]  Marcin Feder,et al.  Structure Prediction and Phylogenetic Analysis of a Functionally Diverse Family of Proteins Homologous to the MT-A70 Subunit of the Human mRNA:m6A Methyltransferase , 2002, Journal of Molecular Evolution.

[42]  I. Kobayashi.  Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. , 2001, Nucleic acids research.

[43]  Janusz M. Bujnicki,et al.  GeneSilico protein structure prediction meta-server , 2003, Nucleic Acids Res..

[44]  A. Plückthun,et al.  Protein folding in the periplasm of Escherichia coli , 1994, Molecular microbiology.

[45]  Michelle G. Giglio,et al.  TIGRFAMs and Genome Properties: tools for the assignment of molecular function and biological process in prokaryotic genomes , 2006, Nucleic Acids Res..

[46]  Marcin Feder,et al.  FRankenstein becomes a cyborg: The automatic recombination and realignment of fold recognition models in CASP6 , 2005, Proteins.

[47]  Michal Boniecki,et al.  Structural bioinformatics analysis of enzymes involved in the biosynthesis pathway of the hypermodified nucleoside ms2io6A37 in tRNA , 2007, Proteins.

[48]  B. Van Houten,et al.  Structural insights into the first incision reaction during nucleotide excision repair , 2005, The EMBO journal.

[49]  Edita Kriukienė,et al.  MnlI--The member of H-N-H subtype of Type IIS restriction endonucleases. , 2005, Biochimica et biophysica acta.

[50]  Leszek Rychlewski,et al.  FFAS03: a server for profile–profile sequence alignments , 2005, Nucleic Acids Res..

[51]  Daniel Fischer,et al.  Twenty thousand ORFan microbial protein families for the biologist? , 2003, Structure.

[52]  D Fischer,et al.  LiveBench‐1: Continuous benchmarking of protein structure prediction servers , 2001, Protein science : a publication of the Protein Society.

[53]  I. Kobayashi,et al.  Restriction-modification systems as genomic parasites in competition for specific sequences. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[54]  R. Huber,et al.  Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues. , 1998, Journal of molecular biology.

[55]  Johannes Söding,et al.  Protein homology detection by HMM?CHMM comparison , 2005, Bioinform..

[56]  Janusz M. Bujnicki,et al.  Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses , 2008, Nucleic acids research.

[57]  J. Bujnicki,et al.  A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy. , 2005, Protein engineering, design & selection : PEDS.

[58]  Marcin Feder,et al.  Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily , 2007, BMC Structural Biology.

[59]  D Fischer,et al.  Hybrid fold recognition: combining sequence derived properties with evolutionary information. , 1999, Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing.

[60]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[61]  T L Blundell,et al.  FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. , 2001, Journal of molecular biology.

[62]  Janusz M. Bujnicki,et al.  MetaMQAP: A meta-server for the quality assessment of protein models , 2008, BMC Bioinformatics.

[63]  M. Blaser,et al.  Purification of the Novel Endonuclease, Hpy188I, and Cloning of Its Restriction-Modification Genes Reveal Evidence of Its Horizontal Transfer to the Helicobacter pylori Genome* , 2000, The Journal of Biological Chemistry.

[64]  J. Bujnicki,et al.  Novel protein fold discovered in the PabI family of restriction enzymes , 2007, Nucleic acids research.

[65]  A. Pingoud,et al.  Type II restriction endonucleases: structure and mechanism , 2005, Cellular and Molecular Life Sciences.

[66]  R. Blumenthal,et al.  Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes. , 1995, Journal of molecular biology.

[67]  I. Kobayashi,et al.  A DNA Methyltransferase Can Protect the Genome from Postdisturbance Attack by a Restriction-Modification Gene Complex , 2002, Journal of bacteriology.

[68]  J Lundström,et al.  Pcons: A neural‐network–based consensus predictor that improves fold recognition , 2001, Protein science : a publication of the Protein Society.

[69]  Janusz M Bujnicki,et al.  Sequence permutations in the molecular evolution of DNA methyltransferases , 2002, BMC Evolutionary Biology.