Abdelghany HM et al. (2003) J Biol Chem "Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis."

McLennan AG, Rafferty JB, Bailey S, Blackburn GM, Abdelghany HM

[J Biol Chem, 2003]

The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap(4)A hydrolase fusion proteins were expressed and their k(cat) and K-m values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k(cat) of 23 s(-1) was reduced by 10(5)-, 10(3)-, and 30-fold, respectively, by replacement of the conserved P-phosphate-binding catalytic residues Glu(56), Glu(52), and Glu(103) by Gln. K-m values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His(31) to Val or Ala and Lys(83) to Met produced 10- and 16-fold increases in K. compared with the wild type value of 8.8 muM. These residues stabilize the P-1-phosphate. H31V and H31A had a normal kcat but K83M showed a 37-fold reduction in k(cat). Lys(36) also stabilizes the P-1-phosphate and a K36M mutant had a 10-fold reduced kcat but a relatively normal K-m. Thus both Lys(36) and Lys(83) may play a role in catalysis. The previously suggested roles of Tyr(27), His(38), Lys(79), and Lys(81) in stabilizing the P-2 and P-3-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr(76) and Tyr(121), which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased K-m 4-fold. It is concluded that interactions with the P-1- and P-4-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that

Abdelghany HM et al. (2001) Biochim Biophys Acta "Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans."

Abdelghany HM, Gasmi L, Cartwright JL, McLennan AG, Bailey S, Rafferty JB

[Biochim Biophys Acta, 2001]

Asymmetrically cleaving diadenosine 5',5(m)-P-1,P-4-tetraphosphate (AP(4)A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coh. 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 Ap(4)A hydrolase. It hydrolyses AP(4)A with a K-m of 7 muM 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 muM) and competitively by adenosine 5'-tetraphosphate with AP(4)A 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 Angstrom and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative AP(4)A 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 AP(4)A hydrolase will help determine the basis of this grouping.

Bailey S et al. (2002) Acta Crystallogr D Biol Crystallogr "Crystallization of a complex of Caenorhabditis elegans diadenosine tetraphosphate hydrolase and a non-hydrolysable substrate analogue, AppCH2ppA."

Rafferty JB, McLennan AG, Abdelghany HM, Blackburn GM, Sedelnikova SE, Bailey S

[Acta Crystallogr D Biol Crystallogr, 2002]

The molecule diadenosine tetraphosphate (Ap(4)A) has been suggested to be a component of the cellular response to metabolic stress and/or, via the intracellular Ap(3)A/Ap(4)A ratio, to be involved in differentiation and apoptosis. Thus, the enzyme Ap(4)A hydrolase has a key metabolic role through regulation of the intracellular Ap(4)A levels. Crystals of this enzyme from the nematode Caenorhabditis elegans have been obtained in the presence of a non-hydrolysable substrate analogue, AppCH(2)ppA. The crystals belong to space group P2(1), unit-cell parameters a = 57.6, b = 36.8, c = 68.9 Angstrom, beta = 114.2degrees, and diffract to approximately 2.0 Angstrom. Determination of the structure of this complex will provide insights into the substrate specificity and catalytic activity of this class of enzymes.

Bailey S et al. (2002) Structure "The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms."

Bailey S, Baker PJ, Abdelghany HM, Blackburn GM, Rafferty JB, Sedelnikova SE, McLennan AG

[Structure, 2002]

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 Angstrom. and 1.8 Angstrom, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.