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

Hend M. Abdelghany, Lakhdar Gasmi, Jared L. Cartwright, Scott Bailey, John B. Rafferty, Alexander G. McLennan

Research output: Contribution to journalArticlepeer-review

Abstract

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 Km of 7 μM and kcat 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 (Ki = 25 μM) and competitively by adenosine 5′-tetraphosphate with Ap4A as substrate (Ki = 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.

Original languageEnglish (US)
Pages (from-to)27-36
Number of pages10
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1550
Issue number1
DOIs
StatePublished - Nov 26 2001
Externally publishedYes

Keywords

  • ApA hydrolase
  • Caenorhabditis elegans
  • Crystallisation
  • Nucleotide
  • Nudix

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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