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- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino 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-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.
Komori
K
K
Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Ichiyanagi
K
K
Morikawa
K
K
Ishino
Y
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D019154:Protein Splicing; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases