dc.contributor |
Barcelona Supercomputing Center |
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.date |
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.citation |
0264-6021 |
dc.identifier.citation |
10.1042/BJ20141211 |
dc.identifier.uri |
http://hdl.handle.net/2117/84134 |
dc.language.iso |
eng |
dc.publisher |
Portland Press |
dc.relation |
http://www.biochemj.org/content/466/2/253 |
dc.relation |
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 |
info:eu-repo/grantAgreement/ES/6PN/BIO2011-26694 |
dc.relation |
info:eu-repo/grantAgreement/ES/1PE/CTQ2013-48287 |
dc.relation |
info:eu-repo/grantAgreement/ES/6PN/BFU2011-24615 |
dc.relation |
info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX |
dc.rights |
Attribution-NonCommercial-NoDerivs 4.0 Spain |
dc.rights |
info:eu-repo/semantics/openAccess |
dc.rights |
http://creativecommons.org/licenses/by-nc-nd/4.0/es/ |
dc.subject |
Àrees temàtiques de la UPC::Enginyeria mecànica::Impacte ambiental |
dc.subject |
Protein |
dc.subject |
Dye-decolorizing peroxidase |
dc.subject |
Site-directed mutagenesis |
dc.subject |
Multifrequency EPR |
dc.subject |
Molecular docking |
dc.subject |
QM/MM |
dc.subject |
Catalytic protein radicals |
dc.subject |
Auricularia auricula-judae |
dc.subject |
Proteïnes |
dc.title |
Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study |
dc.type |
info:eu-repo/semantics/publishedVersion |
dc.type |
info:eu-repo/semantics/article |
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.abstract |
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.description.abstract |
Peer Reviewed |