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 (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. © The Authors Journal compilation © 2015 Biochemical Society.

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 [10.1042/BJ20141211].

Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study

POGNI, REBECCA;BARATTO, MARIA CAMILLA;SINICROPI, ADALGISA;
2015-01-01

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 (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. © The Authors Journal compilation © 2015 Biochemical Society.
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 [10.1042/BJ20141211].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/977022