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 (286)DXD(288) motif and Trp(102), which were shown to be necessary for Mn(2+) and UDP binding, respectively, we identified by alanine scanning Asp(270), Arg(273), Tyr(284), Asn(384), and Trp(520) as being important for enzyme activity. The amino acids Arg(455), Asp(461), Lys(463), and Glu(472) and residues of helix alpha17 (e.g. Glu(449)) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix alpha17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix alpha17 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.
Highlights
The crystal structure of the catalytic domain of toxin B has been solved recently [13] and the structural requirements of the sugar substrate specificity have been determined [14]
We studied the functional role of several amino acids located in or near the active center of toxin B and identified amino acid residues that are involved in protein substrate recognition
Amino Acid Residues Involved in Catalysis and Sugar Substrate Interaction—The glucosyltransferase located in the N-terminal domain of C. difficile belongs to the GT-A family of glycosyltransferases [13]
Summary
Materials—UDP-[14C]glucose (287.4 mCi/mmol) were obtained from PerkinElmer Life Science; PCR primers were from Biochip Technologies (Freiburg, Germany). Toxin B from C. difficile VPI 10463 and lethal toxin (LT) from C. sordellii 6018 and their recombinant catalytic domains were expressed and purified as described previously [14]. Polymerase Chain Reactions—Amplification of the glucosyltransferase domain of toxin B of C. difficile VPI 10463 and the glucosyltransferase domain of C. sordellii 6018 was performed as described previously [5]. Sequencing—All constructs of toxin B and LT fragments were checked by sequencing with ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction kit and ABI 310 Cycle Sequencer (Perkin-Elmer).
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