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

蜡样芽孢杆菌鞘磷脂酶（Bc-SMase）能将鞘磷脂水解成磷碱和神经酰胺，并具有必需的二价金属离子。Bc-SMase是哺乳动物中性SMase （nSMase）的同源酶，可以模拟内源性哺乳动物中性SMase在分化、发育、衰老和凋亡等方面的作用，因此Bc-SMase可能是表征尚不明确的哺乳动物中性SMase的模型。金属离子对Bc-SMase鞘磷脂酶活性的激活顺序为Co^<2+>>=Mn^<2+>>=Mg^<2+>>>Ca^<2+>>=Sr^<2+>。测定了含这些金属离子的Bc-SMase的第一个晶体结构。Co^<2+>和Mg^<2+>的裂口中心的水桥双二价金属离子是发挥鞘磷脂酶活性的催化结构。另一方面，Ca^<2+>结合位点的结构与Ca^<2+>和Mg^<2+>不同。这些金属离子的另一个结合位点在裂缝的一侧边缘。具有Mg^<2+>或Co^<2+>的酶的晶体结构，由于其催化氨基酸残基的共同结构，将为属于DNase I like折叠超家族的磷酸水解酶提供共同的结构基础。此外，结构特征和位点定向诱变表明，具有芳香氨基酸残基的特异性β-发夹参与了与膜SM和底物SM的结合。因此，虽然α -毒素与Bc-SMase溶血的是同一种绵羊红细胞，但其三维结构显然与Bc-SMase不同。从结果来看，毒素诱导溶血的机制似乎与Bc-SMase不同。

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.