title: Identification of Lys-403 in the PI-SceI homing endonuclease as part of a symmetric catalytic center.
abstract: Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed to bind DNA. At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously. Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme. Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity. Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA.
authors.affiliation: Center for Macromolecular Design, Institute of Biosciences and Technology and Department of Biochemistry and Biophysics, Texas A & M University, Houston, Texas 77030, USA. fgimble@ibt.tamu.edu
authors.forename: F S
authors.initials: FS
authors.lastname: Gimble
authors.forename: X
authors.initials: X
authors.lastname: Duan
authors.forename: D
authors.initials: D
authors.lastname: Hu
authors.forename: F A
authors.initials: FA
authors.lastname: Quiocho
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002696:Chlamydomonas / Q000201:enzymology; D015342:DNA Probes / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000032:analysis*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
chemical_list: D015342:DNA Probes; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D008274:Magnesium; D008239:Lysine