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

• Published in: The international journal of biochemistry & cell biology 2010-07, Vol.42 (7), p.1174-1181
• Main Authors: Winward, Lucinda, Whitfield, William G.F., McLennan, Alexander G., Safrany, Stephen T.
• Format: Article
• Language: English
• Subjects: Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - genetics; Acid Anhydride Hydrolases - metabolism; Adenine Nucleotides - metabolism; Amino Acid Sequence; Animals; Cations, Divalent - pharmacology; Chromatography, High Pressure Liquid; Diadenosine polyphosphate; DIPP; Dithiothreitol - pharmacology; Drosophila; Drosophila melanogaster; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; Drosophila Proteins - chemistry; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Embryos; Enzyme Stability - drug effects; Gene Expression Regulation, Developmental - drug effects; Guanosines; Hydrogen-Ion Concentration - drug effects; Hydrolysis; Hydrolysis - drug effects; Inositol diphosphate; Inositol Phosphates - metabolism; Inositols; Kinetics; Molecular Sequence Data; Nudix; Nudix Hydrolases; Organisms; Oxidation-Reduction - drug effects; Polyphosphates; Position (location); Protein Transport - drug effects; Pyrophosphatases - chemistry; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Sequence Alignment; Subcellular Fractions - drug effects; Subcellular Fractions - enzymology; Substrate Specificity - drug effects; Temperature
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/j.biocel.2010.04.003

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 ( K m = 11 μM, k cat = 0.79 s −1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP 5, K m = 0.07 μM, k cat = 0.024 s −1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg 2+ (10–20 mM) or Mn 2+ (1 mM) were preferred for nucleotide hydrolysis and Mg 2+ (0.5–1 mM) or Co 2+ (1–100 μM) for PP-InsP 5 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 H 2O 2 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-InsP 5 to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP 5 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-InsP 5 during development.