Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates.
According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.
Wenz
C
C
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Selent
U
U
Wende
W
W
Jeltsch
A
A
Wolfes
H
H
Pingoud
A
A
D001216:Asparagine / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001216:Asparagine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases