title: A novel endonuclease mechanism directly visualized for I-PpoI.
abstract: A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.
authors.affiliation: Fred Hutchinson Cancer Research Center and the Graduate Programs in Molecular and Cell Biology and Biomolecular Structure and Design, University of Washington, 1100 Fairview Ave. N. A3-023, Seattle, Washington 98109, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Galburt
authors.forename: B
authors.initials: B
authors.lastname: Chevalier
authors.forename: W
authors.initials: W
authors.lastname: Tang
authors.forename: M S
authors.initials: MS
authors.lastname: Jurica
authors.forename: K E
authors.initials: KE
authors.lastname: Flick
authors.forename: R J
authors.initials: RJ
authors.lastname: Monnat
authors.forename: B L
authors.initials: BL
authors.lastname: Stoddard
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002412:Cations / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004583:Electrons; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005583:Fourier Analysis; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D016839:Physarum polycephalum / Q000201:enzymology*; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D012997:Solvents; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D002412:Cations; D010710:Phosphates; D012997:Solvents; D014867:Water; D004247:DNA; D012964:Sodium; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; D008274:Magnesium; D010100:Oxygen