Characterization of auto‐ADP‐ribosylation activity of Cholix toxin

Auto‐ADP‐ribosylation activities have been found among many bacterial and mammalian ADP‐ribosyltransferases. Cholix toxin is an ADP‐ribosyltransferase found in environmental strains of Vibrio cholera. We report here the identification and characterization of the auto‐ADP‐ribosylation activity of the catalytic domain of the toxin. Transfer of ADP‐ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of residues proximal to the NAD+ binding pocket. Site‐directed mutagenesis, two‐dimensional electrophoresis and mass spectrometric analysis show that arginine is the preferred non‐diphthamide acceptor and that there are multiple arginine target residues for auto‐ADP‐ribosylation. These findings are consistent with a reaction mechanism involving a diffusible intermediate in a transition state. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxacarbenium ion, suggesting the intermediate may be a strained form of NAD. Auto‐ADP‐ribosylation is likely to be a SN1‐like reaction proceeding through a diffusible planar intermediate that can generate both [alpha] and[beta]‐anomers of ADP‐ribosylarginine.