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- Ceu I 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- Ceu I 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.
Turmel
M
M
Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada. mturmel@rsvs.ulaval.ca
Otis
C
C
Côté
V
V
Lemieux
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D000460:Chlorophyta / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004247:DNA; D004706:Endodeoxyribonucleases; C071132:endodeoxyribonuclease I-CeuI