Inactivation of the elongation factor Tu by mosquitocidal toxin-catalyzed mono-ADP-ribosylation.
The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an approximately 97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an approximately 45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001407:Bacillus / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001427:Bacterial Toxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C068603:mosquitocidal toxin SSII-1; D000246:Adenosine Diphosphate Ribose; D010444:Peptide Elongation Factor Tu