MUTATIONS AFFECTING THE ACTIVITY OF THE SHIGA-LIKE TOXIN-I A-CHAIN

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eu karyotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been im plicated in catalysis, the crystal structure of Slt-IA has not been so lved nor has the geometry of its active site been well defined. In ord er to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not pre viously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we construct ed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal s tructure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distanc es from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glyco sidase are indeed present on the amino acid side chains occupying the cleft.