Characterization of the catalytic domain of Clostridium novyi alpha-toxin

Clostridium novyi alpha-toxin belongs to the family of large clostridial cy totoxins which act on cells through the modification of small GTP-binding p roteins. We present here an analysis of the catalytic domain of alpha-toxin , A NH2-terminal 551-amino-acid fragment, alpha 551, was found to contain t he full enzyme activity of the holotoxin, whereas a slightly shortened frag ment encompassing 509 amino acids showed no detectable enzyme activity. Fur ther characterization of the enzymatically active fragment alpha 551 reveal ed a substrate specificity for both UDP-N-acetylglucosamine and UDP-glucose , A Michaelis-Menten constant of 17 muM was determined for the substrate UD P-N-acetylglucosamine, while that for UDP-glucose was about 20 times higher , indicating a weaker affinity of the toxin for the latter substrate. Mutat ion of the aspartic acids of a conserved motif DXD within alpha 551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partiall y overcome by increased concentrations of manganese ions, suggesting the in volvement of these aspartic acids in Mn2+ binding. By construction of chime ras of enzymatically active fragments of C, sordellii lethal toxin and C, n ovyi alpha-toxin, we located the region involved in nucleotide-sugar specif icity to amino acids 133 through 517.