title: Crystal structure of the 5hmC specific endonuclease PvuRts1I.
abstract: PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications.
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02109 Warsaw, Poland mbochtler@iimcb.gov.pl.
authors.forename: Asgar Abbas
authors.initials: AA
authors.lastname: Kazrani
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02109 Warsaw, Poland mbochtler@iimcb.gov.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Kowalska
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02109 Warsaw, Poland.
authors.forename: Honorata
authors.initials: H
authors.lastname: Czapinska
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02109 Warsaw, Poland Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02106 Warsaw, Poland mbochtler@iimcb.gov.pl.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bochtler
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D017122:Bacteriophage T4 / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011514:Proteus vulgaris / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004279:DNA, Viral; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004262:DNA Restriction Enzymes; C024369:endodeoxyribonuclease Rts1