USE OF SITE-DIRECTED MUTAGENESIS TO PROBE STRUCTURE-FUNCTION-RELATIONSHIPS OF ALPHA-TOXIN FROM CLOSTRIDIUM-PERFRINGENS

The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cer eus (PC-PLC), for which a high-resolution crystal structure is availab le. This structural information was used as the basis of a site-direct ed mutagenesis strategy in which critical amino acid residues of alpha -toxin involved in zinc binding, interaction with substrate, or cataly sis were replaced. Biochemical studies with the corresponding toxin va riants indicate that there is probably a single active site endowed wi th lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at p osition 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platel et aggregation in vitro.