A Common Motif of Eukaryotic Glycosyltransferases Is Essential for the Enzyme Activity of Large Clostridial Cytotoxins

Abstract

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 Asp 270 and Arg 273 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.

Alternative Splicing in Class V Myosins Determines Association with Rab10

“…Therefore, catalytic activity should play no role in pore formation. This was corroborated by the use of an enzyme deficient mutant (DXD mutant) of recombinant toxin A (39). Increase in rubidium release by this mutant protein indicates that pore formation is functionally independent of glucosyltransferase activity.…”

“…HT-29 cells were preloaded with 86 Rb ϩ and treated either with ToxA, a recombinant toxin A produced in B. megaterium (rToxA), or an enzyme-deficient mutant of toxin A (DXD mutant, produced in B. megaterium, rToxA ed ). The mutation of the aspartic acid residues at positions 286 and 288 to alanine in toxin B was formerly shown to reduce the enzymatic activity by about 1000-fold (39). The corresponding residues in toxin A are at position 285 and 287, and their change to alanine also inhibited glucosyltransferase activity.…”

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity againstClostridium difficileinfection in mice and hamsters

“…41 In addition, trans-membrane domain of TcdB has also been reported to contribute to toxicity. 42 To ensure that mTcd138 was atoxic, 2 amino acids, which have been reported to be the key residues involved in the GT activity, 43,44 were mutated in the GT domain of TcdB (Fig. 1B).…”

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

“…Two highly conserved motifs were chosen for mutation, namely, tryptophan-101 and the DXD motif at positions 285 to 287 (3,17). Analogous mutations of TcdB resulted in reductions of the in vitro GT activity, by factors of 1,000 and 5,000, respectively (5,6,16). The in vitro GT activities of rTcdA (wild type), rTcdA W101A (tryptophan mutant), and rTcdA D285/287N (DXD mutant) were determined from the linear phases of RhoA glucosylation kinetics (Fig.…”