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Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

By Jörg Schirmer, Hans-Joachim Wieden, Marina V. Rodnina and Klaus Aktories

Abstract

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ∼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 ∼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

Topics: Enzymology and Protein Engineering
Publisher: American Society for Microbiology
Year: 2002
DOI identifier: 10.1128/AEM.68.10.4894-4899.2002
OAI identifier: oai:pubmedcentral.nih.gov:126424
Provided by: PubMed Central
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