Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP- ribosylated Ras at multiple sites. One site appeared to be Arg(41), but the second site could not be localized. In this study, the sites of ADP-ribosy lation of c-Ha-Ras by ExoS were directly determined. Under saturating condi tions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose i ncorporated per mol of Ras, Nucleotide occupancy did not influence the stoi chiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg(41) and Arg(128). E xoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Pas, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Has was identified as Arg(135), which was on the same a lpha-helix as Arg(128). Arg(41) and Arg(128) are located within two different secondary structure m otifs, beta-sheet and alpha-helix, respectively, and are spatially separate d within the three-dimensional structure of Has. The fact that ExoS could A DP-ribosylate a target protein at multiple sites, along with earlier observ ations that ExoS could ADP-ribosylate numerous target proteins, were proper ties that have been attributed to several vertebrate ADP-ribosyltransferase s, This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology wit h the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosy ltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ri bosyltransferases.