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dc.contributor.authorLinde, Dolores
dc.contributor.authorPogni, Rebecca
dc.contributor.authorCañellas, Marina
dc.contributor.authorLucas, Fátima
dc.contributor.authorGuallar, Víctor
dc.contributor.authorRuiz-Dueñas, Francisco J.
dc.contributor.authorBaratto, Maria Camilla
dc.contributor.authorSinocro, Adalgisa
dc.contributor.authorCoscolin, Cristina
dc.contributor.authorRomero, Antonio
dc.contributor.authorMedrano, Francisco Javier
dc.contributor.authorMartínez, Angel T.
dc.contributor.otherBarcelona Supercomputing Center
dc.identifier.citationLinde, Dolores [et al.]. Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study. "Biochemical Journal", 01 Març 2015, vol. 466, núm. 2, p. 253-262.
dc.description.abstractDye-decolorizing peroxidase (DyP) of Auricularia auriculajudae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2 activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat>200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19kcat∼20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.
dc.description.sponsorshipWe thank the staff of the SOLEIL (Gyf-sur-Yvette, France) and ALBA (Barcelona, Spain) synchrotrons, and the BSC (Barcelona, Spain) computational facilities. The MALDI– TOF analyses were carried out at the CIB Proteomics facility, a member of the Spanish ProteoRed-ISCIII network.This work was supported by the INDOX [grant number KBBE-2013-7-613549] and PELE [grant number ERC-2009-Adg 25027] European Union projects, by projects of the Spanish Ministry of Economy and Competitiveness (MINECO) [grant number BIO2011-26694, CTQ2013-48287 and BFU2011-24615] and by the Italian Ministry of Education, Universities and Research (MIUR) [project PRIN 2009-STNWX3]. D.L. and F.J.R.-D. are grateful for the financial support of an EU project contract, and a Ramon y Cajal contract of the Spanish Ministry of Economy and Competitiveness (MINECO) respectively.
dc.format.extent10 p.
dc.publisherPortland Press
dc.rightsAttribution-NonCommercial-NoDerivs 4.0 Spain
dc.subjectÀrees temàtiques de la UPC::Enginyeria mecànica::Impacte ambiental
dc.subject.otherDye-decolorizing peroxidase
dc.subject.otherSite-directed mutagenesis
dc.subject.otherMultifrequency EPR
dc.subject.otherMolecular docking
dc.subject.otherCatalytic protein radicals
dc.subject.otherAuricularia auricula-judae
dc.titleCatalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study
dc.description.peerreviewedPeer Reviewed
dc.rights.accessOpen Access
dc.description.versionPostprint (published version)
dc.relation.projectidinfo:eu-repo/grantAgreement/EC/FP7/250277/EU/P.E.L.E (Protein Energy Landscape Exploration): a la carte drug design tools/PELE
dc.relation.projectidinfo:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX
upcommons.citation.publicationNameBiochemical Journal

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