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

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.

Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

“…In this way it is possible to locate and characterize local and global minima structures for the most favorable protein-ligand interactions. PELE has been used successfully in a number of ligand migration studies with both small and large substrates (28)(29)(30). In this work, PELE was set up first to drive the ligands inside the protein until the center of mass was less than 10 Å from the compound I oxygen.…”

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs

“…It has to be pointed out that oxidation of ABTS and anthraquinone dyes (reactive blue series) displayed sigmoidal rate curves (not shown). This "apparent cooperative phenomenon" suggests that TcDyP may have multiple oxidation sites like the fungal DyP from Auricularia auricular-judae (37)(38)(39).…”

“…1A) that has been assigned as the fingerprint of all DyPs (18,23). In plant peroxidases, the arginine within the heme distal area (Arg 38 in HRP) is necessary for peroxidase activity. However, the role of arginine in DyPs (Arg 327 in TcDyP) is still debatable.…”

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

“…Then, following the protocol employed previously to identify catalytically active surface residues in a dye-decolorizing peroxidase [29] , QM/MM calculations were performed on wild-type AaeUPO1. All potential surface oxidation sites (9 tyrosines and 1 tryptophan) were included in the quantum region, where one electron was removed and spin density computed.…”