Nagahama M, Sakurai J
Department of Microbiology, Tokushima Bunri University, Japan.
Microbiol Immunol. 1996;40(3):189-93. doi: 10.1111/j.1348-0421.1996.tb03333.x.
A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.
通过对产气荚膜梭菌α毒素基因进行随机聚合酶链反应（PCR）诱变，分离出一种突变毒素（MT），该毒素几乎消除了α毒素99%的溶血活性。在突变毒素中，Tyr（Y）-62、Thr（T）-74和Ile（I）-345位点的氨基酸分别被His、Ile和Met取代。通过定点诱变将T-74替换为Ile，导致溶血、磷脂酶C和鞘磷脂酶活性丧失至野生型的1/250。单独将Y-62替换为Ile或将I-345替换为Met并不影响毒素的活性。T74I突变体与绵羊红细胞膜结合，并在Tris缓冲盐溶液中以与野生型相同的方式特异性结合[65Zn]2+，每摩尔蛋白质含有2摩尔锌离子。这些结果表明，T-74残基在产气荚膜梭菌α毒素的这些生物学活性中起关键作用。
