title: Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens.
abstract: The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.
authors.affiliation: Unité de Génétique Moléculaire Bactaŕienne, Institut Pasteur, Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Guillouard
authors.forename: T
authors.initials: T
authors.lastname: Garnier
authors.forename: S T
authors.initials: ST
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D001483:Base Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010974:Platelet Aggregation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens