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

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 (Zn2+ or Mg2+). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with Km, kcat and kcat/Km values 9 μM, 43 s−1 and 4.8 μM−1 s−1 (pH 6.5, 0.1 mM Zn2+) and 12 μM, 13 s−1 and 1.1 μM−1 s−1 (pH 7.5, 20 mM Mg2+), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20 mM Mg2+ (Km, kcat and kcat/Km 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 Mg2+ (IC50 = 20 μM), whereas it is ineffective in the presence of Zn2+, supporting the view that inhibition involves a specific, MgF3−-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.