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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Diadenosine oligophosphates are ubiquitous compounds that were discovered over 30 years ago. Diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) is the most studied member of this family, and its function in yeast is unknown. To investigate possible functions, we changed the intracellular Ap(4)A concentration in Schizosaccharomyces pombe via disruption and overexpression of the aph1 gene, which encodes an Ap(4)A hydrolase (Aph1). S. pombe Aph1 is 52% identical with a human tumour suppressor protein, Fhit, in a core region of 109 amino acids. Disruption of aph1 resulted in an 85% decrease in Ap(4)A hydrolase activity and a 290-fold increase in the intracellular Ap(4)A concentration. The disruption and subsequent increase in intracellular Ap(4)A concentration had no significant effect on the growth of S. pombe. Overexpression of the S. pombe aph1 gene, resulting in 17- and 84-fold increases in Ap(4)A hydrolase activity above wild-type levels, resulted in 60 and 80% decreases respectively in the intracellular Ap(4)A concentration. This represents the first report of a decrease in the intracellular Ap(4)A concentration in response to overexpression of a degradative enzyme in any eukaryotic organism. We describe a new S. pombe expression plasmid, pPOX, which was used to achieve the largest increase in expression of aph1. Overexpression of aph1 at the highest level resulted in a 46% increase in generation time in comparison with the control strain. Neither overexpression nor disruption had any effect on the intracellular ATP or ADP concentrations. This is the first report of ADP and ATP concentrations in S. pombe. These data also indicate that Aph1 functions in vivo to degrade Ap(4)A, and that high-level overexpression of this enzyme reduces the growth rate.
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PMID:Disruption and overexpression of the Schizosaccharomyces pombe aph1 gene and the effects on intracellular diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A), ATP and ADP concentrations. 1097 Jul 77

This review concerns enzymes that can degrade nucleoside 5'-tetra- and pentaphosphates (p(4)N and p(5)N) and those that can degrade various dinucleoside polyphosphates (Np(3-6)N'). Most of these enzymes are hydrolases, and they occur in all types of organisms. Certain fungi and protozoa also possess specific Np(n)N' phosphorylases. Specific p(4)N hydrolases have been demonstrated in mammals and in plants. In yeast, p(4)N and p(5)N are hydrolyzed by exopolyphosphatases. Among other hydrolases that can degrade these minor mononucleotides are phosphatases, apyrase, and (asymmetrical) Np(4)N' hydrolase, as well as the nonspecific adenylate deaminase. Np(n)N's are good substrates for Type I phosphodiesterases and nucleotide pyrophosphatases, and diadenosine polyphosphates are easily deaminated to diinosine polyphosphates by nonspecific adenylate deaminases. Specific Np(3)N' hydrolases occur in both prokaryotes and eukaryotes. Interestingly, the human fragile histidine triad (Fhit) tumor suppressor protein appears to be a typical Np(3)N' hydrolase. Among the specific Np(4)N' hydrolases are asymmetrically cleaving ones, which are typical of higher eukaryotes, and symmetrically cleaving enzymes found in Physarum polycephalum and in many bacteria. An enzyme that hydrolyzes both diadenosine tetraphosphate and diadenosine triphosphate has been found in the fission yeast Schizosaccharomyces pombe. Its amino acid sequence is similar to that of the human Fhit/Np(3)N' hydrolase. Very recently, a typical (asymmetrical) Np(4)N' hydrolase has been demonstrated for the first time in a bacterium-the pathogenic Bartonella bacilliformis. Another novelty is the discovery of diadenosine 5', 5"'-P(1),P 6-hexaphosphate hydrolases in budding and fission yeasts and in mammalian cells. These enzymes and the (asymmetrical) Np(4)N' hydrolases have the amino acid motif typical of the MutT (or Nudix hydrolase) family. In contrast, the Schizosaccharomyces pombe Ap(4)A/Ap(3)A hydrolase, the human Fhit protein, and the yeast Np(n)N' phosphorylases belong to a superfamily GAFH, which includes the histidine triad proteins.
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PMID:Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates. 1100 95

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.
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PMID:Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius. 1143 89

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.
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PMID:Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans. 1173 85

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 A, beta = 114.2 degrees, and diffract to approximately 2.0 A. Determination of the structure of this complex will provide insights into the substrate specificity and catalytic activity of this class of enzymes.
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PMID:Crystallization of a complex of Caenorhabditis elegans diadenosine tetraphosphate hydrolase and a non-hydrolysable substrate analogue, AppCH2ppA. 1185 44

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, 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.
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PMID:The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms. 1193 63

Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265-2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine.
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PMID:PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity. 1205 87

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.
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PMID:A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity. 1236 30

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(4)-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(m) compared with the wild type value of 8.8 microm. These residues stabilize the P(1)-phosphate. H31V and H31A had a normal k(cat) 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 k(cat) 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 it may have a much wider substrate range then previously believed.
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PMID:Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis. 1247 70

The gastric pathogen Helicobacter pylori harbors one Nudix hydrolase, NudA, that belongs to the nucleoside polyphosphate hydrolase subgroup. In this work, the enzymatic activity of purified recombinant NudA protein was analyzed on a number of nucleoside polyphosphates. This predicted 18.6-kDa protein preferably hydrolyzes diadenosine tetraphosphate, Ap(4)A at a k(cat) of 0.15 s(-1) and a K(m) of 80 microm, resulting in an asymmetrical cleavage of the molecule into ATP and AMP. To study the biological role of this enzyme in H. pylori, an insertion mutant was constructed. There was a 2-7-fold decrease in survival of the mutant as compared with the wild type after hydrogen peroxide exposure but no difference in survival after heat shock or in spontaneous mutation frequency. Western blot analyses revealed that NudA is constitutively expressed in H. pylori at different growth stages and during stress, which would indicate that this protein has a housekeeping function. Given that H. pylori is a diverse species and that all the H. pylori strains tested in this study harbor the nudA gene and show protein expression, we consider NudA to be an important enzyme in this bacterium.
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PMID:The NudA protein in the gastric pathogen Helicobacter pylori is an ubiquitous and constitutively expressed dinucleoside polyphosphate hydrolase. 1255 7


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