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-...
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Published in | Nucleic acids research Vol. 25; no. 13; pp. 2610 - 2619 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.07.1997
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ark:/67375/HXZ-4947Z8WH-8 istex:0326DD43A76104D97CAEA3AE43970C609C4D1068 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/25.13.2610 |