title: Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis.
abstract: The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Freiburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jank
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Reinert
authors.forename: Torsten
authors.initials: T
authors.lastname: Giesemann
authors.forename: Georg E
authors.initials: GE
authors.lastname: Schulz
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C072292:lethal toxin LT, Clostridium sordellii; C031815:toxB protein, Clostridium difficile; D014537:Uridine Diphosphate N-Acetylglucosamine; D014532:Uridine Diphosphate Glucose