Characterisation of a bis(5′-nucleosyl)-tetraphosphatase (asymmetrical) from Drosophila melanogaster

• Published in: The international journal of biochemistry & cell biology 2007, Vol.39 (5), p.943-954
• Main Authors: Winward, Lucinda, Whitfield, William G.F., Woodman, Timothy J., McLennan, Alexander G., Safrany, Stephen T.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Ap 4A hydrolase; Base Sequence; Cell Nucleus - metabolism; Diadenosine polyphosphate; Dinucleoside Phosphates - metabolism; Drosophila melanogaster; Drosophila melanogaster - embryology; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Enzyme Activation - drug effects; Female; Fluoride; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hydrogen-Ion Concentration; Hydrolysis - drug effects; Kinetics; Magnesium - pharmacology; Microscopy, Fluorescence; Molecular Sequence Data; Nudix; Nudix Hydrolases; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Temperature; Zinc - pharmacology
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/j.biocel.2007.01.017

The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6 kDa Nudix hydrolase (Apf) that specifically metabolizes these nucleotides from a Drosophila melanogaster cDNA. Apf always produces an NTP product, with substrate preference depending on pH and divalent ion (Zn 2+ or Mg 2+). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with K m, k cat and k cat/ K m values 9 μM, 43 s −1 and 4.8 μM −1 s −1 (pH 6.5, 0.1 mM Zn 2+) and 12 μM, 13 s −1 and 1.1 μM −1 s −1 (pH 7.5, 20 mM Mg 2+), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20 mM Mg 2+ ( K m, k cat and k cat/ K m values of 15 μM 4.0 s −1, and 0.27 μM −1 s −1). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg 2+ (IC 50 = 20 μM), whereas it is ineffective in the presence of Zn 2+, supporting the view that inhibition involves a specific, MgF 3 −—containing transition state analogue complex. Patterns of Apf expression in Drosophila tissues show Apf mRNA levels to be highest in embryos and adult females. Subcellular localization with Apf–EGFP fusion constructs reveals Apf to be predominantly nuclear, having an apparent preferential association with euchromatin and facultative heterochromatin. This supports a nuclear function for diadenosine tetraphosphate. Our results show Apf to be a fairly typical member of the bis (5′-nucleosyl)-tetraphosphatase subfamily of Nudix hydrolases with features that distinguish it from a previously reported bis (5′-nucleosyl)-tetraphosphatase hydrolase activity from Drosophila embryos.