Título:
|
Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study
|
Autor/a:
|
Linde, Dolores; Pogni, Rebecca; Cañellas, Marina; Lucas, Fátima; Guallar, Víctor; Ruiz-Dueñas, Francisco J.; Baratto, Maria Camilla; Sinocro, Adalgisa; Coscolin, Cristina; Romero, Antonio; Medrano, Francisco Javier; Martínez, Angel T.
|
Otros autores:
|
Barcelona Supercomputing Center |
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. |
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. |
Abstract:
|
Peer Reviewed |
Materia(s):
|
-Àrees temàtiques de la UPC::Enginyeria mecànica::Impacte ambiental -Protein -Dye-decolorizing peroxidase -Site-directed mutagenesis -Multifrequency EPR -Molecular docking -QM/MM -Catalytic protein radicals -Auricularia auricula-judae -Proteïnes |
Derechos:
|
Attribution-NonCommercial-NoDerivs 4.0 Spain
http://creativecommons.org/licenses/by-nc-nd/4.0/es/ |
Tipo de documento:
|
Artículo - Versión publicada Artículo |
Editor:
|
Portland Press
|
Compartir:
|
|