Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate (Km = 11 μM, kcat = 0.79 s−1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP5, Km = 0.07 μM, kcat = 0.024 s−1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg2+ (10–20 mM) or Mn2+ (1 mM) were preferred for nucleotide hydrolysis and Mg2+ (0.5–1 mM) or Co2+ (1–100 μM) for PP-InsP5 hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H2O2 and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP5 to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP5 found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP5 during development.