Identification of Glu¹⁷³ as the critical amino acid residue for the ADP-ribosyltransferase activity of Clostridium botulinum C3 exoenzyme

Clostridium botulinum C3 exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp¹⁸ and Glu¹⁷³ of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu¹⁷³ 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 Trp¹⁸ remained intact. Swiss 3T3 cells treated with the enzyme with the Trp¹⁸ substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu¹⁷³-substituted enzyme. These results indicate that the Glu¹⁷³ 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.