A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins.
A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.
Busch
C
C
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany.
Hofmann
F
F
Selzer
J
J
Munro
S
S
Jeckel
D
D
Aktories
K
K
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry*; D003013:Clostridium / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D001427:Bacterial Toxins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D008345:Manganese; D005964:Glucosyltransferases; D006867:Hydrolases; D019204:GTP-Binding Proteins; D014532:Uridine Diphosphate Glucose