title: Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis.
abstract: The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.
authors.affiliation: Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Barth
authors.forename: J C
authors.initials: JC
authors.lastname: Preiss
authors.forename: F
authors.initials: F
authors.lastname: Hofmann
authors.forename: K
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D020134:Catalytic Domain; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins; C096324:botulinum toxin type C