Abstract
BackgroundCholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.ResultsOur studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.ConclusionsWe discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-014-0026-1) contains supplementary material, which is available to authorized users.
Highlights
Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera
We showed that wild type cholix toxin catalytic fragment could ADP-ribosylate oligo argininyl peptides and eukaryotic elongation factor 2 (eEF2) (H715R) mutant in which the post-translationally modified diphthamide at His715 was replaced by arginine
Here we show that auto-ADP-ribosylation reaction of cholix toxin primarily involves the intramolecular transfer of ADP-ribosyl moiety to multiple arginine residues located around the NAD+ binding pocket as well as distal sites on the opposite face of the enzyme
Summary
Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. ADP-ribosyltransferases (ADPRTs) constitute a broadly distributed family of prokaryotic and eukaryotic enzymes that catalyze the transfer of an ADP-ribose moiety from NAD+ to one or more substrates. A number of ADPRTs exhibit auto-ADP-ribosylation activity, including diphtheria toxin [17,18], cholera toxin [19], Clostridium limosum C3 exoenzyme [20], Pseudomonas aeruginosa exoenzyme S [21], Escherichia coli heat-labile enterotoxin [22], Neisseria meningitidis ADP-ribosylargininyl transferase, NarE [23], and several mammalian ADPRTs [24,25,26]. Auto-ADP-ribosylation of mammalian ADP-ribosyltransferase 5 (ART5) redirects the enzyme catalytic focus from NAD+ glycohydrolase activity to ADPribosyltransferase activity [28]. Auto-ADP-ribosylation has been shown to inhibit the activity of the C. limosum C3 exoenzyme, P. aeruginosa exoenzyme S, and Neisseria meningitides ADP-ribosylargininyl transferase, NarE [20,21,23]
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