Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (Phospholipase C) which are required for its biological activities

A panel of random mutants within the DNA encoding the carboxy-terminal doma in of Clostridium perfringens alpha -toxin was constructed. Three mutants w ere identified which encoded cu-toxin variants (Lys330Glu, Asp305Gly, and A sp293Ser) with reduced hemolytic activity. These variants also had diminish ed phospholipase C activity toward aggregated egg yolk phospholipid and red uced cytotoxic and myotoxic activities. Asp305Gly showed a significantly in creased enzymatic activity toward the monodisperse substrate rho NPPC, wher eas Asp293Ser displayed a reduced activity toward this phospholipid analogu e. In addition, Asp293Ser showed an increased dependence on calcium for enz ymatic activity toward aggregated phospholipid and appeared calcium-deplete d in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly s howed altered dependence on calcium for enzymatic activity toward aggregate d phospholipid. Asp305 is located in the interface between the amino- and c arboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed resi dues which may play a role in the recognition of membrane phospholipids. (C ) 2000 Academic Press.