Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.
AUTOR(ES)
Hovde, C J
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=280038Documentos Relacionados
- Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.
- Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity.
- Development and evaluation of enzyme-linked immunosorbent assays for detection of shiga-like toxin I and shiga-like toxin II.
- Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR.
- Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.