Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease

• Published in: Nucleic acids research Vol. 25; no. 13; pp. 2610 - 2619
• Main Authors: Turmel, Monique, Otis, Christian, Côté, Vincent, Lemieux, Claude
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.07.1997
• Subjects: Amino Acid Sequence; Base Sequence; Chlorophyta - enzymology; Chloroplasts - enzymology; Conserved Sequence; DNA - metabolism; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Evolution, Molecular; Introns; Kinetics; Molecular Sequence Data; Mutation; Sequence Alignment; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/25.13.2610

Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I-CeuI is lethal in Escherichia coli, enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis.