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

Lucinda Winward, William G. F. Whitfield, Timothy J Woodman, Alexander G. McLennan, Stephen T. Safrany

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6kDa 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 9microM, 43s(-1) and 4.8microM(-1)s(-1) (pH 6.5, 0.1mMZn(2+)) and 12microM, 13s(-1) and 1.1microM(-1)s(-1) (pH 7.5, 20mMMg(2+)), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20mMMg(2+) (K(m), k(cat) and k(cat)/K(m) values of 15microM 4.0s(-1), and 0.27microM(-1)s(-1)). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg(2+) (IC(50)=20microM), 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.
    Original languageEnglish
    Pages (from-to)943-954
    Number of pages12
    JournalInternational Journal of Biochemistry & Cell Biology
    Volume39
    Issue number5
    DOIs
    Publication statusPublished - 2007

    Keywords

    • Adenosine Triphosphate
    • Amino Acid Sequence
    • Animals
    • Base Sequence
    • Cell Nucleus
    • Dinucleoside Phosphates
    • Drosophila Proteins
    • Drosophila melanogaster
    • Enzyme Activation
    • Female
    • Gene Expression Regulation, Developmental
    • Gene Expression Regulation, Enzymologic
    • Green Fluorescent Proteins
    • Hydrogen-Ion Concentration
    • Hydrolysis
    • Kinetics
    • Magnesium
    • Microscopy, Fluorescence
    • Molecular Sequence Data
    • Pyrophosphatases
    • Recombinant Fusion Proteins
    • Reverse Transcriptase Polymerase Chain Reaction
    • Sequence Homology, Amino Acid
    • Temperature
    • Zinc

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