Evidence that Arg‐295, Glu‐378, and Glu‐380 are active‐site residues of the ADP‐ribosyltransferase activity of iota toxin

The active site of the enzymatic component (Ia) of the Clostridium perfringens iota toxin has been studied by site-directed mutagenesis. Sequence alignment showed that Ia and C3 enzymes display a segment in their C-terminal part which is homologous to that forming the active domain of pertussis toxin, cholera toxin, and Escherichia coli thermolabile toxins. This structure consists of a beta-strand and an alpha-helix which forms the NAD-binding cavity and which is flanked by two catalytic spatially conserved residues involved in catalysis [Domenighini et al. (1994) Mol. Microbiol. 14, 41-50]. Substitutions (Arg-295-Lys, Glu-378-Ala, Glu-380-Asp, and Glu-380-Ala) induced a drastic decrease in ADP-ribosylation and cytotoxic activities, while substitution of the adjacent Arg (Arg-296-Lys) only partially affected the enzymatic activity and cytotoxicity. These results indicate that Arg-295, Glu-378 and Glu-380 of Ia are involved in the ADP-ribosylation activity which is essential for the morphological changes of cells treated with iota toxin.