期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1988
卷号：85
期号：8
页码：2568-2572
DOI：10.1073/pnas.85.8.2568
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
