Structure and function relationship of diarrheagenic toxins produced by enteric bacteria

采用定点诱变方法研究了肠出血性大肠埃希菌产生的Vero毒素与副溶血性弧菌产生的耐热性直接溶血素的结构和功能关系。Vero毒素具有RNA n -糖苷酶活性，可在动物细胞60S核糖体亚基28S核糖体RNA 5′端4324位切割腺苷糖苷键。这种酶活性的水解导致EF-1依赖的氨基酰基trna与60S核糖体亚基的结合受到抑制，从而抑制蛋白质合成。突变体Vero毒素E167Q （n端167位谷氨酸被谷氨酰胺取代）、R170L和E167Q-R170L的酶活性和生物活性均显著降低，表明167位谷氨酸和170位精氨酸对Vero毒素的活性起重要作用。W65H、W65Y、W65L和L66S等突变体毒素均表现出较低的生物活性，表明n端65位的Trp和66位的Leu对各种生物活性起着重要作用。

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.