Identification of Residues in the Carboxy-Terminal Domain of Clostridium perfringens α-Toxin (Phospholipase C) Which Are Required for Its Biological Activities (original) (raw)

2000, Archives of Biochemistry and Biophysics

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens ␣-toxin was constructed. Three mutants were identified which encoded ␣-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate NPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the aminoand carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.