Molecular biological and structural analysis of toxic and enzymatic activities in Clostridium perfringens alpha-toxin

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Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyeiin to phosphocoline and ceramide with the essential divalent metal ion. Bc-SMase is a homologous enzyme of mammalian neutral SMase (nSMase), and mimics the action of the endogenous mammalian nSMase in causing differentiation, development, aging and apoptosis, thus Bc-SMase would be a model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order of Co^<2+>>=Mn^<2+>>=Mg^<2+>>>Ca^<2+>>=Sr^<2+>. The first crystal structures of Bc-SMase with these metal ions were determined. The water bridged double divalent metal ions at the center of cleft in both of Co^<2+> and Mg^<2+> were concluded to be the catalytic architecture to exert the sphingomyelinase activities. On the other hands, the architecture of Ca^<2+> binding at the site was different from that of Ca^<2+> and Mg^<2+>. There is the other binding site of these metal ions at one side edge of the cleft. The crystal structure of the enzyme with Mg^<2+> or Co^<2+> would provide the common structure basis among phosphohydrolases belonging DNase I like folding superfamily, due to their common architecture of the catalytic amino acid residues. In addition, the structural features and site directed mutagenesis suggest that the specific β-hairpin with the aromatic amino acid residues participates in binding to membrane SM and the substrate SM. Therefore, It is apparent that the 3D structure of alpha-toxin is different from that of Bc-SMase, although the toxin hemolyzes the same sheep erythrocytes as Bc-SMase. From the results, the mechanism of hemolysis induced by the toxin seems to be is different from that of Bc-SMase.