Guillouard I, Garnier T, Cole S T
Unité de Génétique Moléculaire Bactaŕienne, Institut Pasteur, Paris, France.
Infect Immun. 1996 Jul;64(7):2440-4. doi: 10.1128/iai.64.7.2440-2444.1996.
The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.
产气荚膜梭菌α毒素的氨基末端结构域与蜡样芽孢杆菌的完整磷脂酶C（PC-PLC）高度同源，蜡样芽孢杆菌的完整磷脂酶C已有高分辨率晶体结构。该结构信息被用作定点诱变策略的基础，在该策略中，参与锌结合、与底物相互作用或催化的α毒素关键氨基酸残基被替换。对相应毒素变体的生化研究表明，可能存在一个具有卵磷脂酶、鞘磷脂酶和溶血活性的单一活性位点。通过使用一种高度纯化的变体，其中第56位的催化天冬氨酸残基被天冬酰胺取代，结果表明磷脂酶活性对于体内致死性和体外介导血小板聚集至关重要。
