Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study
Tipus de documentArticle
Condicions d'accésAccés obert
Projecte de la Comissió EuropeaPELE - P.E.L.E (Protein Energy Landscape Exploration): a la carte drug design tools (EC-FP7-250277)
INDOX - Optimized oxidoreductases for medium and large scale industrial biotransformations (EC-FP7-613549)
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 (RB19kcat∼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.
CitacióLinde, Dolores [et al.]. Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study. "Biochemical Journal", 01 Març 2015, vol. 466, núm. 2, p. 253-262.
Versió de l'editorhttp://www.biochemj.org/content/466/2/253