Title: Identification of Glu<sup>173</sup> as the critical amino acid residue for the ADP‐ribosyltransferase activity of <i>Clostridium botulinum</i> C3 exoenzyme
Abstract: Clostridium botulinum C3 exoenzyme specifically ADP‐ribosylates rho‐p21 in eukaryotic cells. Trp 18 and Glu 173 of this enzyme were substituted with other amino acids via site‐directed mutagenesis. All substitutions at Glu 173 caused a significant reduction in affinity for NAD and diminished ADP‐ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp 18 remained intact. Swiss 3T3 cells treated with the enzyme with the Trp 18 substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu 173 ‐substituted enzyme. These results indicate that the Glu 173 residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP‐ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.