Structure and function relationship of diarrheagenic toxins produced by enteric bacteria

用定点突变方法研究了肠出血性大肠杆菌产生的Vero毒素和副溶血弧菌产生的耐热直接溶血素的结构与功能关系，Vero毒素具有RNA N-糖苷酶活性，能裂解动物细胞60 S核糖体亚基28 S核糖体RNA 5'端4324位的腺苷糖苷键。E167 Q等Vero细胞毒素的酶活性和生物学活性的研究表明，E167 Q等Vero细胞毒素的酶活性和生物学活性均高于E167 Q，而E167 Q等Vero细胞毒素的酶活性和生物学活性均低于E167 Q（从N-末端开始的位置167处的谷氨酸被谷氨酰胺替代）、R170 L和E167 Q-R170 L，对副溶血性弧菌（Vibrio parahaemolyticus）的耐热直接溶血素W 65 H、W 65 Y、W 65 H、W 65 Y、W 65 W 65 L和L 66 S显示出显著低的生物活性，表明N-末端第65位的Trp和第66位的Leu对于各种生物活性是重要的。

Structure and function relationship of Vero toxin produced by enterohemorrhagic Escherichia coli and of thermostable direct hemolysin produced by Vibrio parahaemolyticus was studied by using site-directed mutagenesis method.Vero toxin has an enzymatic activity of RNA N-glycosidase and cleaves a glycosidic bond of adeninosine at 4324th position from 5' terminus of 28S ribosomal RNA of 60S ribosomal subunit in animal cells. Hydrolysis by this enzymatic activity results in the inhibition of EF-1 dependent aminoacyl-tRNA binding to 60S ribosomal subunit, which inhibition of protein synthesis.The enzymatic as well as biological activity of mutant Vero toxins, such as E167Q (glutamic acid at position 167 from N-terminus is replaced by glutamine), R170L and E167Q-R170L, were significantly decreased indicating that glutamic acid at position 167 and arginine at position 170 are important for the activity of Vero toxin.Site-directed mutagenesis was also applied to the thermostable direct hemolysin of Vibrio parahaemolyticus Mutant toxins, such as W65H, W65Y, W65L and L66S showed significantly low biological activity indicating that Trp at position 65 from N-terminus and Leu at position 66 were important for the various biological activities.