Figure 5. Activity of NUDT15 towards potential NUDIX family substrates.

(a) Substrate hydrolysis after 30 min incubation at 22 °C was monitored by coupling the reaction to an excess of bovine alkaline phosphatase or E. coli pyrophosphatase and measuring inorganic phosphate using malachite green reagent. Data are presented as v (hydrolysed substrate (μM) per minute) per [NUDT15] (μM). Depicted data are mean±s.d. of triplicates. Experiment was performed twice with similar results. (b) Relative activity of NUDT15 by HPLC analysis. Activity of 10 nM NUDT15 protein against a panel of potential NUDIX substrates was measured by HPLC after 30 min incubation at 37 °C. Enzymatic activity was stopped by addition of trifluoroacetic acid (5%) and samples were ran under conditions indicated in Methods section. Per cent hydrolysis was calculated by subtracting the peak area at 30 min from the area at time 0 and dividing this by the area at time 0; (n=2). (c) Substrate hydrolysis after 20 min incubation at 22 °C with NUDT15 (black bars) or MTH1 (white bars). Substrate hydrolysis was monitored by coupling the reaction to phosphatase or pyrophosphatase and measuring the presence of inorganic phosphate as described above. Experiments were performed in duplicate and data are presented as v (hydrolysed substrate (μM) per minute) per [enzyme] (μM). The experiment was repeated with similar results. (d) Mutation of NUDT15 (cyan) Arg139 to Cys is implicated in thiopurine-induced leukopaenia. Arg139 is located at the base of the helix α2 adjacent to Cys140. The NUDIX box is shown in magenta.