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 per fringens iota toxin has been studied by site-directed mutagenesis. Seq uence alignment showed that Ia and C3 enzymes display a segment in the ir C-terminal part which is homologous to that forming the active doma in of pertussis toxin, cholera toxin, and Escherichia coli thermolabil e toxins, This structure consists of a beta-strand and an alpha-helix which forms the NAD-binding cavity and which is flanked by two catalyt ic 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 a djacent Arg (Arg-296-Lys) only partially affected the enzymatic activi ty and cytotoxicity, These results indicate that Arg-295, Glu-378 and Glu-380 of Ia are involved in the ADP-ribosylation activity which is e ssential for the morphological changes of cells treated with iota toxi n.