PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization of the binding and cleavage abilities by site-directed mutagenesis

PI-Pful and PI-Pfull from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by pr otein splicing from the precursor protein including ribonucleotide reductas e (RNR), We show here that both enzymes specifically interact with their su bstrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respe ctively, They have two copies of the am ino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specifi c mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI-Pful, and Asp156 and Asp249 in PI-Pfull, were critical fo r catalysis, The third residues of the active site triads, as predicted fro m the structure of PI-SceI, were Asn225 in PI-Pful and Lys224 in PI-Pfull. Substitution of Asn225 in PI-Pful by Ala did not affect catalysis, The clea vage activity of PI-Pfull was 50-fold decreased by the substitution of Ala for Lys224, The binding affinity of the mutant protein for the substrate DN A also decreased B-fold, The Lys in PI-Pfull may play a direct or indirect role in catalysis of the endonuclease activity.