Conformational Changes and Reaction of Clostridial Glycosylating Toxins

Abstract

The crystal structures of the catalytic fragments of ‘lethal toxin’ from Clostridium sordellii and of ‘α-toxin’ from Clostridium novyi have been established. Almost half of the residues follow the chain fold of the glycosyl-transferase type A family of enzymes; the other half forms large α-helical protrusions that are likely to confer specificity for the respective targeted subgroup of Rho proteins in the cell. In the crystal, the active center of α-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn 2 + , was catalytically competent. Surprisingly, the structure of lethal toxin with Ca 2 + (instead of Mn 2 + ) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn 2 + is indispensable to the integrity of the enzyme. The homologous structures of α-toxin without substrate, distorted lethal toxin with Ca 2 + plus donor, active lethal toxin with Mn 2 + plus donor and the homologous Clostridium difficile toxin B with a hydrolyzed donor have been lined up to show the geometry of several reaction steps. Interestingly, the structural refinement of one of the three crystallographically independent molecules of Ca 2 + -ligated lethal toxin resulted in the glucosyl half-chair conformation expected for glycosyl-transferases that retain the anomeric configuration at the C1″ atom. A superposition of six acceptor substrates bound to homologous enzymes yielded the position of the nucleophilic acceptor atom with a deviation of < 1 Å. The resulting donor–acceptor geometry suggests that the reaction runs as a circular electron transfer in a six-membered ring, which involves the deprotonation of the nucleophile by the β-phosphoryl group of the donor substrate UDP-glucose.