Walker N, Holley J, Naylor C E, Flores-Díaz M, Alape-Girón A, Carter G, Carr F J, Thelestam M, Keyte M, Moss D S, Basak A K, Miller J, Titball R W
Defence Evaluation and Research Agency, Salisbury, Wilts, United Kingdom.
Arch Biochem Biophys. 2000 Dec 1;384(1):24-30. doi: 10.1006/abbi.2000.2065.
A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.
构建了一组编码产气荚膜梭菌α毒素羧基末端结构域的DNA随机突变体。鉴定出三个突变体，它们编码溶血活性降低的α毒素变体（Lys330Glu、Asp305Gly和Asp293Ser）。这些变体对聚集的蛋黄磷脂的磷脂酶C活性也降低，细胞毒性和肌毒性活性也降低。Asp305Gly对单分散底物rhoNPPC的酶活性显著增加，而Asp293Ser对这种磷脂类似物的活性降低。此外，Asp293Ser对聚集磷脂的酶活性表现出对钙的依赖性增加，并且在PAGE带移分析中显示钙缺乏。相比之下，Lys330Glu和Asp305Gly对聚集磷脂的酶活性对钙的依赖性均未改变。Asp305位于氨基末端和羧基末端结构域之间的界面，而Asp293和Lys330是表面暴露的残基，可能在膜磷脂的识别中起作用。
