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Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study
dc.contributor.author | Linde, Dolores |
dc.contributor.author | Pogni, Rebecca |
dc.contributor.author | Cañellas, Marina |
dc.contributor.author | Lucas, Fátima |
dc.contributor.author | Guallar, Víctor |
dc.contributor.author | Ruiz-Dueñas, Francisco J. |
dc.contributor.author | Baratto, Maria Camilla |
dc.contributor.author | Sinocro, Adalgisa |
dc.contributor.author | Coscolin, Cristina |
dc.contributor.author | Romero, Antonio |
dc.contributor.author | Medrano, Francisco Javier |
dc.contributor.author | Martínez, Angel T. |
dc.contributor.other | Barcelona Supercomputing Center |
dc.date.accessioned | 2016-03-10T12:05:30Z |
dc.date.available | 2016-03-10T12:05:30Z |
dc.date.issued | 2015-03-01 |
dc.identifier.citation | Linde, 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.identifier.issn | 0264-6021 |
dc.identifier.uri | http://hdl.handle.net/2117/84134 |
dc.description.abstract | Dye-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.sponsorship | We 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.extent | 10 p. |
dc.language.iso | eng |
dc.publisher | Portland Press |
dc.rights | Attribution-NonCommercial-NoDerivs 4.0 Spain |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/es/ |
dc.subject | Àrees temàtiques de la UPC::Enginyeria mecànica::Impacte ambiental |
dc.subject.lcsh | Protein |
dc.subject.other | Dye-decolorizing peroxidase |
dc.subject.other | Site-directed mutagenesis |
dc.subject.other | Multifrequency EPR |
dc.subject.other | Molecular docking |
dc.subject.other | QM/MM |
dc.subject.other | Catalytic protein radicals |
dc.subject.other | Auricularia auricula-judae |
dc.title | Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study |
dc.type | Article |
dc.subject.lemac | Proteïnes |
dc.identifier.doi | 10.1042/BJ20141211 |
dc.description.peerreviewed | Peer Reviewed |
dc.relation.publisherversion | http://www.biochemj.org/content/466/2/253 |
dc.rights.access | Open Access |
dc.description.version | Postprint (published version) |
dc.relation.projectid | info:eu-repo/grantAgreement/EC/FP7/250277/EU/P.E.L.E (Protein Energy Landscape Exploration): a la carte drug design tools/PELE |
dc.relation.projectid | info:eu-repo/grantAgreement/MICINN//BIO2011-26694/ES/BUSQUEDA E INGENIERIA DE NUEVAS PEROXIDASAS FUNGICAS DE ALTO POTENCIAL REDOX/ |
dc.relation.projectid | info:eu-repo/grantAgreement/MINECO//CTQ2013-48287-R/ES/DISENYO COMPUTACIONAL RACIONAL DE OXIDOREDUCTASAS PARA APLICACIONES INDUSTRIALES Y TECNOLOGICAS/ |
dc.relation.projectid | info:eu-repo/grantAgreement/MICINN//BFU2011-24615/ES/ANALISIS ESTRUCTURAL DE LOS PRINCIPALES MECANISMOS DE RESISTENCIA ANTIBIOTICA Y VIRULENCIA BACTERIANA: DESDE LAS PROTEINAS DE LA PARED CELULAR A LOS SISTEMAS DE SECRECION/ |
dc.relation.projectid | info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX |
local.citation.publicationName | Biochemical Journal |
local.citation.volume | 466 |
local.citation.number | 2 |
local.citation.startingPage | 253 |
local.citation.endingPage | 262 |
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