title: Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis.
abstract: The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same.
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: J
authors.initials: J
authors.lastname: Yukitake
authors.forename: Y
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: A
authors.initials: A
authors.lastname: Miyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity; D001483:Base Sequence; D001681:Biological Assay; D003545:Cysteine; D004220:Disulfides; D004352:Drug Resistance, Microbial; D004768:Enterotoxins / Q000235:genetics* / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D051379:Mice; D009154:Mutation; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); D013752:Tetracycline; D003545:Cysteine