title: Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin.
abstract: A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.
authors.affiliation: Department of Microbiology, Tokushima Bunri University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nagahama
authors.forename: J
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003016:Clostridium perfringens / Q000235:genetics*; D006461:Hemolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase