Structure and Substrate-binding Mechanism of Human Ap4A Hydrolase

Abstract

Asymmetric diadenosine 5,5ٟ-P 1 ,P 4 -tetraphosphate (Ap 4 A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap 4 A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap 4 A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.Nudix (nucleoside diphosphate linked to x) hydrolases are a superfamily of enzymes with over 300 identified members (1) that are required for maintaining physiological homeostasis by the metabolism of signaling molecules and potentially toxic substances. This superfamily is subdivided according to substrate specificity, including dinucleotide polyphosphatases (including Ap 4 A 1 hydrolases), ribo-or deoxyribonucleoside triphosphatases, and pyrophosphatases of nucleotide sugars, NADH, and ADP-ribose. All of the Nudix enzyme structures solved to date display the same "prototypical" scaffold comprising an ␣-␤-␣ sandwich fold in which one of the helices and the loop preceding it make up the catalytic site for metal-mediated hydrolysis of the substrate. This loop-helix motif contains the generalized "Nudix signature," GX 5 EX 7 REUXEEXGU, in which U is a bulky aliphatic residue usually Ile, Leu, or Val. Although the folds are similar, loop and helix insertions are commonly observed outside of the highly conserved catalytic region. As a consequence, the family displays a range of nucleotide-binding positions even for similar substrates (2). Furthermore, dimeric enzymes are also observed in which the substrate can be found between adjacent monomers. Perhaps the most striking example of this to date is in the x-ray structure of ADP-ribose pyrophosphatase (3), which requires dimerization through extensive domain swapping for substrate recognition and catalytic activity. Hence, throughout evolution, nature has preserved the scaffold of the reaction sub-site and facilitated diverse substrate recognition by employing residue or loop insertions and in some cases adopted contributions from each monomer in the dimeric state.Nudix Ap 4 A hydrolases (hereafter referred to as Ap 4 Aases) have been isolated from all kingdoms of life (1). Phylogenetic analysis (4) further reveals two distinct groups within these kingdoms. Plant and bacterial enzymes fall ...

Molecular Characterization and Mutational Analysis of Recombinant Diadenosine 5′,5″-P¹,P⁴-Tetraphosphate Hydrolase from <i>Plasmodium falciparum</i>

“…However, less attention has been focused on residues involved in substrate binding. 25) The aromatic residues, which stack one substrate adenosine moiety between them, are highly conserved in structure-based amino acid sequence alignments of animal and plant Ap 4 A hydrolases, 17) suggesting that both residues have important effects on substrate binding. The importance of stacking residues for substrate binding in C. elegans has been previously confirmed by replacing Tyr121 by Ala, which led to an 8-fold increase in wild-type K m value.…”

“…Homology Modeling A comparison of the three dimensional structures of human (as observed in the NMR structure) 17) and P. falciparum (as predicted from protein modeling) Ap 4 A hydrolases indicated an overall similarity between both enzymes (data not shown), with an interesting difference at the adenosine binding site. In the human enzyme, the adenosine moiety is stacked between two phenyl rings of Tyr87 and Phe133 leaving a small space, while the adenosine moiety of the enzyme of P. falciparum lies between the phenyl ring of Tyr87 on one side and Pro133 on other side, leaving a larger space (Fig.…”

A phase II study of sequential neoadjuvant gemcitabine plus doxorubicin followed by gemcitabine plus cisplatin in patients with operable breast cancer: prediction of response using molecular profiling

“…According to EASE, the GO categories 'nucleotide/nucleic acid metabolism,' 'RNA metabolism,' 'DNA repair,' and 'response to stress' were overrepresented relative to chance in the gene set highly expressed in tumours with CR at cycle 8 (Figure 2, bottom gene dendrogram branch). Interestingly, the complete responders at cycle 8 showed high expression of NUDT2, which is not only involved in nucleotide metabolism but may also promote apoptosis (Vartanian Swarbrick et al, 2005). Supplementary Figure 2 shows the hierarchical clustering of tumours using the 66-gene set predictive of overall CR (88% accuracy in the 10-fold CV analysis).…”

Diadenosine Tetraphosphate Hydrolase Is Part of the Transcriptional Regulation Network in Immunologically Activated Mast Cells

“…The Nudix type 2 (nudt2) gene product, Ap 4 A hydrolase, has been isolated and cloned and its structure has been determined (33). This enzyme hydrolyzes Ap 4 A into AMP and ATP.…”