Please use this identifier to cite or link to this item: http://hdl.handle.net/20.500.12779/6510
Title: Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study
Authors: Dolores LINDE
Pogni, Rebecca 
Marina CANELLAS
Fátima LUCAS
Victor GUALLAR
Baratto, Maria Camilla 
Sinicropi, Adalgisa 
Verónica SÁEZ-JIMÉNEZ
Cristina COSCOLÍN
Antonio ROMERO
Francisco Javier MEDRANO
Francisco J. RUIZ-DUEÑAS
Angel T. MARTÍNEZ
Keywords: dye-decolorizing peroxidase, site-directed mutagenesis, EPR spectroscopy, molecular docking, QM/MM, catalytic protein radicals
Issue Date: 2015
Project: None 
Journal: BIOCHEMICAL JOURNAL
Abstract: 
Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in E. coli, as a representative for new DyP family, and subjected to mutagenesis, 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 software provided several binding-energy optima for the anthraquinone-type Reactive Blue 19 (RB19) near the above aromatic residues, and the haem access-channel. Subsequent QM/MM calculations showed a higher tendency of Trp-377 than other exposed haem-neighbour residues to harbour a catalytic protein radical, and identified the electrontransfer 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 (RB19 kcat ~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.
Description: 
71842
URI: http://hdl.handle.net/20.500.12779/6510
ISSN: 0264-6021
DOI: 10.1042/BJ20141211
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