Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.
Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-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 microM 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 microM) 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 A and belong to either space group C222 or C222(1). 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.
Abdelghany
H M
HM
School of Biological Sciences, University of Liverpool, UK.
Gasmi
L
L
Cartwright
J L
JL
Bailey
S
S
Rafferty
J B
JB
McLennan
A G
AG
D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D018076:DNA, Complementary / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D014961:X-Ray Diffraction
D018076:DNA, Complementary; D017766:Acid Anhydride Hydrolases; C036849:bis(5'-nucleosyl)tetraphosphatase (symmetrical)