Biochemical journal, 2015-03, Vol.466 (2), p.253-262
2015
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- Autor(en) / Beteiligte
- Titel
- Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study
- Ist Teil von
- Biochemical journal, 2015-03, Vol.466 (2), p.253-262
- Ort / Verlag
- England: Portland Press Ltd
- Erscheinungsjahr
- 2015
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- 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 (k(cat) > 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 k(cat) ~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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0264-6021eISSN: 1470-8728DOI: 10.1042/bj20141211Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4357238
- Format
- –
- Schlagworte
- Amino Acid Substitution, Basidiomycota - enzymology, Binding Sites, Biocatalysis, Coloring Agents - chemistry, Coloring Agents - metabolism, Free Radicals - chemistry, Fungal Proteins - chemistry, Fungal Proteins - genetics, Fungal Proteins - metabolism, Hemeproteins - chemistry, Hemeproteins - genetics, Hemeproteins - metabolism, Models, Molecular, Molecular Docking Simulation, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Mutant Proteins - chemistry, Mutant Proteins - metabolism, Oxidation-Reduction, Peroxidases - chemistry, Peroxidases - genetics, Peroxidases - metabolism, Protein Conformation, Recombinant Proteins - chemistry, Recombinant Proteins - metabolism, Surface Properties, Tryptophan - chemistry, Tyrosine - chemistry