Conformational changes and reaction of clostridial glycosylating toxins.
The crystal structures of the catalytic fragments of 'lethal toxin' from Clostridium sordellii and of 'alpha-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 alpha-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 alpha-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn2+, was catalytically competent. Surprisingly, the structure of lethal toxin with Ca2+ (instead of Mn2+) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn2+ is indispensable to the integrity of the enzyme. The homologous structures of alpha-toxin without substrate, distorted lethal toxin with Ca2+ plus donor, active lethal toxin with Mn2+ 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 Ca2+-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 A. 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 beta-phosphoryl group of the donor substrate UDP-glucose.
Ziegler
Mathias O P
MO
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, D-79104 Freiburg im Breisgau, Germany.
Jank
Thomas
T
Aktories
Klaus
K
Schulz
Georg E
GE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006031:Glycosylation; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile; D008345:Manganese; D002118:Calcium