Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding

Abstract

Recently the crystal structure of the catalytic domain of Clostridium difficile toxin B was solved (Reinert, D. J., Jank, T., Aktories, K., and Schulz, G. E. (2005) J. Mol. Biol. 351, 973-981). On the basis of this structure, we studied the functional role of several amino acids located in the catalytic center of toxin B. Besides the 286DXD288 motif and Trp102, which were shown to be necessary for Mn2+ and UDP binding, respectively, we identified by alanine scanning Asp270, Arg273, Tyr284, Asn384, and Trp520 as being important for enzyme activity. The amino acids Arg455, Asp461, Lys463, and Glu472 and residues of helix α17 (e.g. Glu449) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix α17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix α17 is involved in RhoA recognition by toxin B. The data allow the design of a model of the interaction of the glucosyltransferase domain of toxin B with its protein substrate RhoA. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.