A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

• Published in: The Journal of biological chemistry Vol. 265; no. 36; pp. 22520 - 22525
• Main Authors: Tsuji, T, Inoue, T, Miyama, A, Okamoto, K, Honda, T, Miwatani, T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.12.1990; American Society for Biochemistry and Molecular Biology
• Subjects: amino acid composition; Amino Acid Sequence; Animals; Bacterial Toxins - genetics; Bacterial Toxins - isolation & purification; Bacterial Toxins - toxicity; Bacteriology; Base Sequence; Biological and medical sciences; Capillary Permeability - drug effects; detoxification; enterotoxins; Enterotoxins - genetics; Enterotoxins - isolation & purification; Enterotoxins - toxicity; Escherichia coli - genetics; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Macromolecular Substances; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Rabbits; Recombinant Proteins - isolation & purification; Recombinant Proteins - toxicity; Sequence Homology, Nucleic Acid; Skin - blood supply; Characterization; Plasmid; Proteins; Structure activity relation; Escherichia coli; Active site; Point mutation; Enterotoxin; Bacteria; Cytotoxicity; Chemical composition; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)45736-5

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.