title: Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.
abstract: Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nagahama
authors.forename: Y
authors.initials: Y
authors.lastname: Okagawa
authors.forename: T
authors.initials: T
authors.lastname: Nakayama
authors.forename: E
authors.initials: E
authors.lastname: Nishioka
authors.forename: J
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent / Q000378:metabolism; D003016:Clostridium perfringens / Q000235:genetics*; D004910:Erythrocyte Membrane / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000633:toxicity; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000235:genetics; D015032:Zinc / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D006639:Histidine; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D015032:Zinc