Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

Discovery of inhibitors of lupin diadenosine 5',5'''-P(1),P(4)-tetraphosphate hydrolase by virtual screening.

Novel inhibitors of lupin diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolase have been identified by in silico screening of a large virtual chemical library. Compounds were ranked on the basis of a consensus from six scoring functions. From the top 100 ranked compounds six were selected and initially screened for inhibitory activity using a single concentration isothermal titration calorimetry assay. Two of these compounds that showed excellent solubility properties were further analyzed, but only one [NSC51531; 2-((8-hydroxy-4-(4-methyl-2-sulfoanilino)-9,10-dioxo-9,10-dihydro-1-anthracenyl)amino)-5-methylbenzenesulfonic acid] exhibited competitive inhibition with a K(i) of 1 microM. A structural analogue of this compound also exhibited competitive inhibition with a comparable K(i) of 2.9 microM. (1)H, (15)N NMR spectroscopy was used to map the binding site of NSC51531 on lupin Ap(4)A hydrolase and demonstrated that the compound bound specifically in the substrate-binding site, consistent with the competitive inhibition results. Binding of NSC51531 to the human form of Ap(4)A hydrolase is nonspecific, suggesting that this compound may represent a useful lead in the design of specific inhibitors of the plant-like form of Ap(4)A hydrolases.

The structure of Ap(4)A hydrolase complexed with ATP-MgF(x) reveals the basis of substrate binding.

Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.

Structure and substrate-binding mechanism of human Ap4A hydrolase.

Asymmetric diadenosine 5',5'''-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.