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.
Perelle
S
S
Unité des Toxines Microbiennes, Institut Pasteur, Paris, France.
Domenighini
M
M
Popoff
M R
MR
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D003014:Clostridium botulinum; D003016:Clostridium perfringens / Q000201:enzymology*; D004926:Escherichia coli; D005260:Female; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D001427:Bacterial Toxins; D011994:Recombinant Proteins; C050737:iota toxin, Clostridium perfringens; D018698:Glutamic Acid; D001120:Arginine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases