title: Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities.
abstract: Toxins A and B of Clostridium difficile are UDP-glucose glucosyltransferases that exert their cellular toxicity primarily through their abilities to monoglucosylate, and thereby inactivate, Rho family small GTPases. Toxin A also hydrolyzes UDP-glucose, although this activity is not well characterized. In this study, we measured the kinetics of UDP-glucose hydrolysis by toxins A and B and found significant differences in the catalytic activities of these two structurally homologous toxins. The toxins displayed similar Michaelis constants (Km) for UDP-glucose, but the maximal velocity (Vmax) of toxin B was approximately 5-fold greater than that of toxin A. Toxins A and B exert their enzymatic actions intracellularly, and, interestingly, we found that each toxin absolutely required K+ for optimal hydrolase activity; Na+ was inactive. The toxins also required certain divalent cations for activity and exhibited a significantly greater Vmax and lower Km in the presence of Mn2+ as compared with Mg2+. We conclude that C. difficile toxins A and B are cation-dependent UDP-glucose hydrolases that differ significantly in their catalytic activities, a finding that may have important implications in understanding their different cytotoxic effects.
authors.affiliation: Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: W P
authors.initials: WP
authors.lastname: Ciesla
authors.forename: D A
authors.initials: DA
authors.lastname: Bobak
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D002384:Catalysis; D002412:Cations; D016360:Clostridioides difficile; D004768:Enterotoxins / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002412:Cations; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D008345:Manganese; D005964:Glucosyltransferases; D008274:Magnesium; D011188:Potassium