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J Biol Chem, Vol. 273, Issue 46, 30524-30529, November 13, 1998

Identification of Lys-403 in the PI-SceI Homing Endonuclease as Part of a Symmetric Catalytic Center

Frederick S. Gimble, Xiaoqun Duan^¶, Dongli Hu, and Florante A. Quiocho^¶

From the  Center for Macromolecular Design, Institute of Biosciences and Technology and Department of Biochemistry and Biophysics, Texas A & M University and the ^¶ Graduate Program in Structural and Computational Biology and Molecular Biophysics, Howard Hughes Medical Institute and Verna Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030

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.

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