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dc.contributor.authorHernández-Ortega, Aitor-
dc.contributor.authorBorrelli, Kenneth-
dc.contributor.authorFerreira, Patricia-
dc.contributor.authorMedina, Milagros-
dc.contributor.authorMartínez, Ángel T.-
dc.contributor.authorGuallar, Victor-
dc.date.accessioned2012-05-04T11:43:16Z-
dc.date.available2012-05-04T11:43:16Z-
dc.date.issued2011-03-04-
dc.identifier.citationBiochemical Journal 436: 341-350(2011)es_ES
dc.identifier.issn0264-6021-
dc.identifier.urihttp://hdl.handle.net/10261/49233-
dc.description10 páginas, 7 figuras, 2 tablas -- PAGS nros. 341-350es_ES
dc.description.abstractAAO (aryl-alcohol oxidase) provides H2O2 in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr92. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His502 (and CHO His466), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAOes_ES
dc.language.isoenges_ES
dc.publisherPortland Presses_ES
dc.rightsopenAccesses_ES
dc.subjectAryl-alcohol oxidasees_ES
dc.subjectCatalytic basees_ES
dc.subjectglucose-methanol-choline oxidase (GMC) oxidoreductasees_ES
dc.subjectmolecular dockinges_ES
dc.subjectquantum mechanics/molecular mechanics (QM/MM)es_ES
dc.subjectreaction mechanismes_ES
dc.titleSubstrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidasees_ES
dc.typeartículoes_ES
dc.identifier.doi10.1042/BJ20102090-
dc.description.peerreviewedPeer reviewedes_ES
dc.relation.publisherversionhttp://dx.doi.org/10.1042/BJ20102090es_ES
dc.identifier.e-issn1470-8728-
dc.embargo.terms2012-03-04es_ES
dc.type.coarhttp://purl.org/coar/resource_type/c_6501es_ES
item.openairetypeartículo-
item.cerifentitytypePublications-
item.fulltextNo Fulltext-
item.languageiso639-1en-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.grantfulltextnone-
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