Characterization of Vis Toxin, a Novel ADP-Ribosyltransferase from Vibrio splendidus

Vis toxin was identified by a bioinformatics strategy as a putative virulence factor produced by Vibrio splendidus with mono-ADP-ribosyltransferase activity. Vis was purified to homogeneity as a 28 kDa single-domain enzyme and was shown to possess NADtglycohydrolase [Km(NAD+) = 276 +/- 12 mu M] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [K-M(agmatine) = 272 +/- 18 mM]. Mass spectrometry analysis revealed that Vis labels 1,-arginine with ADP-ribose from the NAD(+) substrate at the amino nitrogen of the guanidinium side chain. Vis is toxic to yeast when expressed in the cytoplasm under control of the CUP1 promotor, and catalytic variants lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. Several small molecule inhibitors were identified from a virtual screen, and the most potent compounds were found to inhibit the transferase activity of the enzyme with K-i values ranging from 25 to 134 mu M. Inhibitor compound M6 bears the necessary attributes of a solid candidate as a lead compound for therapeutic development. Vis toxin was crystallized, and the structures of the apoenzyme (1.4 A) and the enzyme bound with NAD(+) (1.8 angstrom) and with the M6 inhibitor (1.5 angstrom) were determined. The structures revealed that Vis represents a new subgroup within the mono-ADP-ribosyltransferase toxin family.