Cloning, characterisation and crystallisation of a diadenosine 5′,5‴-P¹,P⁴-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans

Asymmetrically cleaving diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap₄A hydrolase. It hydrolyses Ap₄A with a K_m of 7 μM and k_cat of 27 s^-1 producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K_i = 25 μM) and competitively by adenosine 5′-tetraphosphate with Ap₄A as substrate (K_i = 10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 Å and belong to either space group C222 or C222₁. Phylogenetic analysis of known and putative Ap₄A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap₄A hydrolase will help determine the basis of this grouping.