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

蜡状芽孢杆菌的鞘磷脂酶(BC-sMase)与必需的二价金属离子一起将鞘磷脂水解成磷胆碱和神经酰胺。BC-sMase是哺乳动物中性sMase(NSMase)的同源酶，它模拟了哺乳动物内源性nSMase在诱导分化、发育、衰老和凋亡过程中的作用，因此BC-sMase可能是一个功能较差的哺乳动物nSMase的模型。金属离子对BC-sMase鞘磷脂酶活性的激活顺序为Co^&lt；2+&gt；&gt；=Mn^&lt；2+&gt；&gt；=Mg^&lt；2+&gt；&gt；&gt；Ca^&lt；2+&gt；&gt；=Sr^&lt；2+&gt；.测定了这些金属离子与BC-sMase的第一次晶体结构。Co^&lt；2+&gt；和Mg^&lt；2+&gt；裂隙中心的水桥连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，结合部位的Ca^&lt；2+&gt；不同于Ca^&lt；2+&gt；和Mg^&lt；2+&gt；在缝隙的一侧边缘有这些金属离子的另一个结合部位。由于具有催化氨基酸残基的共性结构，该酶的晶体结构可能为属于DNase I类折叠超家族的磷酸水解酶提供了共同的结构基础。此外，结构特征和定点突变表明，带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，尽管α-毒素与BC-sMase溶血的绵羊红细胞相同，但α-毒素的三维结构明显不同于BC-sMase。结果表明，该毒素的溶血机制可能与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.