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.
|Titolo:||Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study|
|Citazione:||Dolores, L., Pogni, R., Marina, C., Fátima, L., Victor, G., Baratto, M.C., et al. (2015). Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study. BIOCHEMICAL JOURNAL, 466(2), 253-262.|
|Appare nelle tipologie:||1.1 Articolo in rivista|
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