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Bifunctional Homodimeric Triokinase/FMN Cyclase

2014Journal article Open access
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dc.contributor.authorRodrigues, Joaquim Rui
dc.contributor.authorCouto, Ana
dc.contributor.authorCabezas, Alicia
dc.contributor.authorPinto, Rosa María
dc.contributor.authorRibeiro, João Meireles
dc.contributor.authorCanales, José
dc.contributor.authorCostas, María Jesús
dc.contributor.authorCameselle, José Carlos
dc.date.accessioned2018-02-12T16:22:05Z
dc.date.available2018-02-12T16:22:05Z
dc.date.issued2014
dc.description.abstractMammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1074/jbc.M113.525626pt_PT
dc.identifier.issn1083-351X
dc.identifier.urihttp://hdl.handle.net/10400.8/3027
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.subjectAnimalspt_PT
dc.subjectCatalysispt_PT
dc.subjectCatalytic Domainpt_PT
dc.subjectDimerizationpt_PT
dc.subjectFlavin-Adenine Dinucleotidept_PT
dc.subjectFructosept_PT
dc.subjectGlyceraldehydept_PT
dc.subjectHumanspt_PT
dc.subjectHydrogen-Ion Concentrationpt_PT
dc.subjectKineticspt_PT
dc.subjectMolecular Dynamics Simulationpt_PT
dc.subjectMutagenesis, Site-Directedpt_PT
dc.subjectMutationpt_PT
dc.subjectPhosphorus-Oxygen Lyasespt_PT
dc.subjectPhosphorylationpt_PT
dc.subjectPhosphotransferases (Alcohol Group Acceptor)pt_PT
dc.subjectProtein Structure, Tertiarypt_PT
dc.subjectRecombinant Proteinspt_PT
dc.subjectSubstrate Specificitypt_PT
dc.titleBifunctional Homodimeric Triokinase/FMN Cyclasept_PT
dc.title.alternativeContribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movementspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage10636pt_PT
oaire.citation.issue15pt_PT
oaire.citation.startPage10620pt_PT
oaire.citation.titleJournal of Biological Chemistrypt_PT
oaire.citation.volume289pt_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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