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Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase

2018-01-18Journal article Open access
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dc.contributor.authorRibeiro, João Meireles
dc.contributor.authorCanales, José
dc.contributor.authorCabezas, Alicia
dc.contributor.authorRodrigues, Joaquim Rui
dc.contributor.authorPinto, Rosa María
dc.contributor.authorLópez-Villamizar, Iralis
dc.contributor.authorCostas, María Jesús
dc.contributor.authorCameselle, José Carlos
dc.date.accessioned2018-02-07T10:13:10Z
dc.date.available2018-02-07T10:13:10Z
dc.date.issued2018-01-18
dc.description.abstractCyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the ‘northern’ ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the ‘northern’ ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR kcat/KM ≈20–200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doiDOI:10.1038/s41598-017-18393-9pt_PT
dc.identifier.issn2045-2322
dc.identifier.urihttp://hdl.handle.net/10400.8/3007
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.relation.publisherversionhttp://rdcu.be/Gpwept_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectAssay systemspt_PT
dc.subjectBiocatalysispt_PT
dc.subjectCalcium signallingpt_PT
dc.subjectEnzyme mechanismspt_PT
dc.subjectProtein designpt_PT
dc.titleSpecific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatasept_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.startPage1036pt_PT
oaire.citation.titleScientific Reportspt_PT
oaire.citation.volume8pt_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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