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 (K(m)=11 microM, 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 microM, k(cat)=0.024 s(-1)). Assayed at 26 degrees 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 microM) 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 degrees C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 degrees C. Heat lability was restored by H(2)O(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.
Winward
Lucinda
L
Division of Cell Signalling, University of Dundee, Dow Street, Dundee DD1 4HN, United Kingdom. lusi83@hotmail.com
Whitfield
William G F
WG
McLennan
Alexander G
AG
Safrany
Stephen T
ST
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D000096964:Nudix Hydrolases
D017766:Acid Anhydride Hydrolases; D000227:Adenine Nucleotides; D002413:Cations, Divalent; D004229:Dithiothreitol; D029721:Drosophila Proteins; D007295:Inositol Phosphates; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C079849:1-diphosphoinositol pentakisphosphate; C552307:Aps protein, Drosophila; C116368:diphosphoinositol polyphosphate phosphohydrolase