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

Kayoko Komori, Kenji Ichiyanagi, Kosuke Morikawa, Yoshizumi Ishino

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

PI-PfuI and PI-PfuII 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°and 67°, 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-PfuI, and Asp156 and Asp249 in PI-PfuII, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI-SceI, were Asn225 in PI-PfuI and Lys224 in PI-PfuII. Substitution of Asn225 in PI-PfuI by Ala did not affect catalysis. The cleavage activity of PI-PfuII 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-PfuII may play a direct or indirect role in catalysis of the endonuclease activity.

Original languageEnglish
Pages (from-to)4175-4182
Number of pages8
JournalNucleic acids research
Volume27
Issue number21
DOIs
Publication statusPublished - Nov 1 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

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