title: Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities.
abstract: The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nagahama
authors.forename: T
authors.initials: T
authors.lastname: Nakayama
authors.forename: K
authors.initials: K
authors.lastname: Michiue
authors.forename: J
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000472:pathogenicity*; D006461:Hemolysis / Q000187:drug effects*; D016297:Mutagenesis, Site-Directed; D012756:Sheep; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000032:analysis
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C040649:alpha toxin, Clostridium perfringens; D015032:Zinc