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

Abstract

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 NAD(+)-glycohydrolase [KM(NAD(+)) = 276 ± 12 μM] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [KM(agmatine) = 272 ± 18 mM]. Mass spectrometry analysis revealed that Vis labels l-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 Ki values ranging from 25 to 134 μM. 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 Å) and the enzyme bound with NAD(+) (1.8 Å) and with the M6 inhibitor (1.5 Å) were determined. The structures revealed that Vis represents a new subgroup within the mono-ADP-ribosyltransferase toxin family.

Characterization of the toxin Plx2A, a RhoA‐targeting ADP‐ribosyltransferase produced by the honey bee pathogen Paenibacillus larvae

“…The thermal stability of Plx2A wt and Plx2A variants was checked in triplicate measurements by melting curve analysis in a StepOnePlus Real‐time PCR system (Applied Biosystems, Foster City, CA, USA) by the use of Protein thermal shift dye according to the manufacturer's instructions (Applied Biosystems, Waltham, MA, USA) according to our earlier method (Ravulapalli et al ., ). Melting curve analysis of the purified proteins established that Plx2A wt and all Plx2A variants each had one distinct melting point and similar melting temperatures (Table ) indicating that all purified proteins were stable and properly folded.…”

Characterization of C3larvinA, a novel RhoA-targeting ADP-ribosyltransferase toxin produced by the honey bee pathogen,Paenibacillus larvae

“…The thermal stability of C3larvinA WT and variants was measured in triplicate measurements by melting-curve analysis in a StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, U.S.A.) using Protein Thermal Shift dye, Sypro Orange ® according to the manufacturer's instructions (Applied Biosystems) adopted from a previous method [46,47]. Melting curve analysis of the purified proteins established that C3larvinA and all active-site variants had a single, distinct melting point and similar melting temperature values (T M ) (Table 1) indicating that all purified proteins were stable and properly folded.…”

14-3-3 proteins activatePseudomonasexotoxins-S and –T by chaperoning a hydrophobic surface

“…The structure was solved by molecular replacement using PDB entries 2c23 (14-3-3) and 4xzj (Vis toxin) as search model. 21,29 The resulting structure ( Supplementary Table 1) served as molecular replacement template for all following datasets.…”