Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases.
Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection. Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine. Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin. From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.
Busch
C
C
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, Freiburg D-79104, Germany.
Hofmann
F
F
Gerhard
R
R
Aktories
K
K
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016360:Clostridioides difficile / Q000378:metabolism*; D017124:Conserved Sequence; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D014364:Tryptophan; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D001427:Bacterial Toxins; D014364:Tryptophan; D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose