Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding.
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.
Jank
Thomas
T
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany.
Giesemann
Torsten
T
Aktories
Klaus
K
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016360:Clostridioides difficile / Q000378:metabolism*; D047989:Paraclostridium sordellii / Q000378:metabolism; D005786:Gene Expression Regulation; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry
D000596:Amino Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C072292:lethal toxin LT, Clostridium sordellii; C031815:toxB protein, Clostridium difficile; D008345:Manganese; D000409:Alanine