title: Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.
abstract: Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.
mesh_terms: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid; D001427:Bacterial Toxins; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D005971:Glutamates; D018698:Glutamic Acid; D007739:L Cells / Q000166:cytology / Q000187:drug effects; D051379:Mice; D009154:Mutation; D010430:Pentosyltransferases / Q000378:metabolism; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001224:Aspartic Acid; D001427:Bacterial Toxins; D005098:Exotoxins; D005971:Glutamates; D018698:Glutamic Acid; D010430:Pentosyltransferases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A