title: Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.
abstract: The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 μM, K(m)(actin) = 24 μM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 μM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Adin
authors.initials: A
authors.lastname: Shniffer
authors.forename: Danielle D
authors.initials: DD
authors.lastname: Visschedyk
authors.forename: Ravikiran
authors.initials: R
authors.lastname: Ravulapalli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Suarez
authors.forename: Zachari J
authors.initials: ZJ
authors.lastname: Turgeon
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Petrie
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Chopra
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000737:chemistry*; D016980:Aeromonas hydrophila / Q000201:enzymology*; D020816:Amino Acid Motifs; D017209:Apoptosis; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D048430:Cell Shape; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D013499:Surface Properties
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D036002:ADP Ribose Transferases