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

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 K-m and k(cat) 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.