The peptide demodification assay was described earlier47 (link). Briefly, peptide concentration for the assay were estimated using absorbance at λ260nm with a molar extinction coefficient of 13,400 M−1 cm−1 for the ADP-ribosyl modification. 8 μM of indicated peptide was demodified by incubation with 1 μM indicated hydrolase (or various concentrations of MavL/Larg1) for 30 min at 30 °C in assay buffer (50 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.15 μM human NUDT5)65 (link). Reactions were stopped and analyzed by performing the AMP-Glo™ assay (Promega) according to the manufacturer’s protocol. Luminescence was recorded on a SpectraMax M5 plate reader (Molecular Devices) and data analyzed with GraphPad Prism 7. For background subtraction reaction were carried out in absence of hydrolase.
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