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.