Nucleic Acids Research, Vol 25, Issue 13 2610-2619, Copyright © 1997 by Oxford University Press
M Turmel, C Otis, V Cote and C Lemieux
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.
ARTICLES
Evolutionarily conserved and functionally important residues in the I- CeuI homing endonuclease
Program in Evolutionary Biology, Canadian Institute for Advanced Research, Departement de Biochimie, Faculte des Sciences et de Genie, Universite Laval, Quebec, Quebec G1K 7P4, Canada. mturmel@rsvs.ulaval.ca
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