Abstract
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.
Highlights
The family of large clostridial cytotoxins consists of the toxins A and B of Clostridium difficile, the lethal and hemorrhagic toxins of Clostridium sordellii, and the ␣-toxin from Clostridium novyi [1, 2]
We report on a tryptophan residue located in this second conserved region of many putative glycosyltransferases, including clostridial cytotoxins, which is likely to be implicated in the binding of the cosubstrate UDP-glucose
Besides the DXD motif, which we found to be essential for enzyme activity, this entire region contains 16 additional residues conserved among all large clostridial cytotoxins (Fig. 1A)
Summary
Materials—14C-Labeled UDP-hexoses were obtained from NEN Life Science Products (Dreieich, Germany). Polymerase chain reaction primers were from MWG Biotech (Ebersberg, Germany). All other reagents were of analytical grade and purchased from commercial sources. Polymerase Chain Reaction Amplification—Amplification of NH2terminal C. difficile toxin B and C. sordellii lethal toxin fragments and construction of COOH-terminal truncated fragments B5461 and LT546 was done as described previously [15, 16]. Site-directed Mutagenesis of Toxin Fragment LT546 and B546 —The QuikChange KitTM (Stratagene) was used for mutating one or two nucleotides in the pGEX 2T-LT546 construct or in the pGEX 2T-B546 construct, respectively. Procedures were carried out according to the manufacturer’s instructions. Primers were constructed as follows: K96A.LT546: Primer pair S1K96A sense/antisense (5Ј-CTGCTGCTTCTGCTATATTACGAATAT-3Ј/5Ј-GATATTCGTAATATAGCAGAAGCAGCA-3Ј)
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