title: Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment.
abstract: In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1-3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.
authors.affiliation: Institut für medizinische Mikrobiologie und Hygiene, Johannes Gutenberg-Universität, Mainz, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Wagenknecht-Wiesner
authors.forename: M
authors.initials: M
authors.lastname: Weidmann
authors.forename: V
authors.initials: V
authors.lastname: Braun
authors.forename: P
authors.initials: P
authors.lastname: Leukel
authors.forename: M
authors.initials: M
authors.lastname: Moos
authors.forename: C
authors.initials: C
authors.lastname: von Eichel-Streiber
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases