Importance of arginine at position 170 of the A subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia coli for toxin activity.
Comparison of the primary structures of the A subunits of Vero toxin 1 (VT1), Vero toxin 2 (VT2), and two variants of VT2 (VT2vp and VT2vh) and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1). All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis. To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis. A total of 22 mutants were prepared to examine 14 conserved residues, and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1. Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically. These results suggest that, in addition to the glutamic acid at position 167 reported previously, arginine at position 170 also plays an important role in the toxin activity of VT1. A possible chemical mechanism of the enzymatic (N-glycosidase) activity of VT1 is proposed based on the relative activities of various mutants.
Yamasaki
S
S
Department of Microbiology, Faculty of Medicine, Kyoto University, Japan.
Furutani
M
M
Ito
K
K
Igarashi
K
K
Nishibuchi
M
M
Takeda
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001483:Base Sequence; D001665:Binding Sites; D016923:Cell Death; D003470:Culture Media; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D022622:Shiga Toxin 1; D014709:Vero Cells
D001427:Bacterial Toxins; D003470:Culture Media; D003603:Cytotoxins; D022622:Shiga Toxin 1; D001120:Arginine