Faculty Collaboration Database

Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases. J Biol Chem 1999 Apr 02;274(14):9503-8

Date

03/27/1999

Pubmed ID

10092634

DOI

10.1074/jbc.274.14.9503

Scopus ID

2-s2.0-0033515484 (requires institutional sign-in at Scopus site)   33 Citations

Abstract

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry 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 Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.

Author List

Ganesan AK, Mende-Mueller L, Selzer J, Barbieri JT

Author

Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

ADP Ribose Transferases
Amino Acid Sequence
Arginine
Bacterial Toxins
Chromatography, High Pressure Liquid
Mass Spectrometry
Models, Molecular
Molecular Sequence Data
Protein Conformation
Protein Structure, Secondary
Pseudomonas aeruginosa
Sequence Alignment
Structure-Activity Relationship
ras Proteins