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Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

2015Journal article Open access
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dc.contributor.authorCabezas, Alicia
dc.contributor.authorRibeiro, João Meireles
dc.contributor.authorRodrigues, Joaquim Rui
dc.contributor.authorLópez-Villamizar, Iralis
dc.contributor.authorFernández, Ascensión
dc.contributor.authorCanales, José
dc.contributor.authorPinto, Rosa María
dc.contributor.authorCostas, María Jesús
dc.contributor.authorCameselle, José Carlos
dc.date.accessioned2018-02-07T10:14:53Z
dc.date.available2018-02-07T10:14:53Z
dc.date.issued2015
dc.description.abstractAmong metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1371/journal.pone.0118680pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.8/3009
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectAcid Anhydride Hydrolasespt_PT
dc.subjectAdenosine Diphosphate Ribosept_PT
dc.subjectAnimalspt_PT
dc.subjectApyrasept_PT
dc.subjectCatalytic Domainpt_PT
dc.subjectHumanspt_PT
dc.subjectLiverpt_PT
dc.subjectManganesept_PT
dc.subjectModels, Molecularpt_PT
dc.subjectMolecular Docking Simulationpt_PT
dc.subjectMolecular Sequence Datapt_PT
dc.subjectMutagenesis, Site-Directedpt_PT
dc.subjectRatspt_PT
dc.subjectStructural Homology, Proteinpt_PT
dc.titleMolecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolasept_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.issue2pt_PT
oaire.citation.startPagee0118680pt_PT
oaire.citation.titlePLOS ONEpt_PT
oaire.citation.volume10pt_PT
person.familyNameRodrigues
person.givenNameJoaquim Rui
person.identifier.ciencia-id9018-0B83-E2C6
person.identifier.orcid0000-0002-9756-1124
person.identifier.ridL-4137-2014
person.identifier.scopus-author-id10242931100
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublication52f6ffb2-43e9-4f78-b414-dbac46f80305
relation.isAuthorOfPublication.latestForDiscovery52f6ffb2-43e9-4f78-b414-dbac46f80305

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