Characterization of the enzymatic component of Clostridium perfringens iota-toxin.
The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.
Nagahama
M
M
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
Sakaguchi
Y
Y
Kobayashi
K
K
Ochi
S
S
Sakurai
J
J
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D020134:Catalytic Domain; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
D001427:Bacterial Toxins; C050737:iota toxin, Clostridium perfringens; D009243:NAD; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase