title: Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.
abstract: Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E. coli. Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid. As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA. Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase. Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation. We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.
authors: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
authors: C J
authors: CJ
authors: Hovde
authors: S B
authors: SB
authors: Calderwood
authors: J J
authors: JJ
authors: Mekalanos
authors: R J
authors: RJ
authors: Collier
mesh_terms: D001427:Bacterial Toxins; D001665:Binding Sites; D004252:DNA Mutational Analysis; D005971:Glutamates; D010446:Peptide Fragments / Q000032:analysis; D011500:Protein Synthesis Inhibitors; D012276:Ricin; D022622:Shiga Toxin 1; D014357:Trypsin
chemical_list: D001427:Bacterial Toxins; D005971:Glutamates; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; D022622:Shiga Toxin 1; D012276:Ricin; D014357:Trypsin