1. Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study
- Author
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Ángel T. Martínez, Rebecca Pogni, Francisco J. Medrano, Marina Cañellas, Dolores Linde, Victor Guallar, Verónica Sáez-Jiménez, Francisco J. Ruiz-Dueñas, Adalgisa Sinicropi, Antonio A. Romero, Cristina Coscolín, Maria Camilla Baratto, Fátima Lucas, and Barcelona Supercomputing Center
- Subjects
Models, Molecular ,Protein Conformation ,ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) ,VP, versatile peroxidase ,dye-decolorizing peroxidase, site-directed mutagenesis, EPR spectroscopy, molecular docking, QM/MM, catalytic protein radicals ,QM, quantum mechanics ,VA, veratryl alcohol ,Multifrequency EPR ,Biochemistry ,Protein structure ,catalytic protein radicals ,Auricularia auricula-judae ,NBS, N-bromosuccinimide ,Coloring Agents ,Site-directed mutagenesis ,Dye decolorizing peroxidase ,chemistry.chemical_classification ,Fungal protein ,biology ,Chemistry ,Enginyeria mecànica::Impacte ambiental [Àrees temàtiques de la UPC] ,Tryptophan ,PELE, Protein Energy Landscape Exploration ,Recombinant Proteins ,hfcc, hyperfine coupling constant ,Molecular Docking Simulation ,Peroxidases ,catalytic protein radical ,Molecular docking ,site-directed mutagenesis ,Oxidation-Reduction ,Research Article ,EPR spectroscopy ,RB19, Reactive Blue 19 ,Peroxidase ,Hemeproteins ,Free Radicals ,Surface Properties ,Stereochemistry ,LRET, long-range electron transfer ,DyP, dye-decolorizing peroxidase ,Molecular Dynamics Simulation ,QM/MM ,Fungal Proteins ,RB5, Reactive Black 5 ,Oxidoreductase ,dye-decolorizing peroxidase ,Enzyme kinetics ,LiP, lignin peroxidase ,Catalytic protein radicals ,Molecular Biology ,Dye-decolorizing peroxidase ,Binding Sites ,Basidiomycota ,Protein ,DMP, 2,6-dimethoxyphenol ,MM, molecular mechanics ,molecular docking ,Cell Biology ,WT, wild-type ,TNM, tetranitromethane ,Amino Acid Substitution ,Biocatalysis ,Mutagenesis, Site-Directed ,biology.protein ,Tyrosine ,Mutant Proteins ,Proteïnes - Abstract
Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae 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 (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., We demonstrate that an exposed tryptophan is responsible for high-turnover oxidation by DyP, a representative of a new protein superfamily. Long-range electron transfer from surface tryptophan residues forming radicals appears as a general mechanism for peroxidase oxidation of bulky substrates.
- Published
- 2015