Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

Abstract

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in H2O2 activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds (J. Biol. Chem. 2015, 290, 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.

Enantioselective Synthesis of Dilignol Model Compounds and Their Stereodiscrimination Study with a Dye-Decolorizing Peroxidase

“…42, 43 Additionally, this 34-residue loop is close to W263, which has been characterized as a surface-exposed substrate oxidation site in Tc DyP. 40 The closest distance between the loop (L279) and W263 is 6.1 Å. Moreover, this loop is widely present in DyPs although the size varies between classes.…”

Rational design of heme enzymes for biodegradation of pollutants toward a green future

“…These Tyr/Trp residues in the secondary sphere of heme, particularly Trp263, play crucial roles in supporting the dye-decolorizing activity of TcDyP. Lu and coworkers [47] showed that replacement of Trp263 with Phe/Ser/Ala resulted in ß40% loss of the activity, with mutation of other residues (Trp158/Trp194/Trp237) loss of ß15% (Fig. 1B).…”

“…(B) Effects of the residual mutation on the activity of TcDyP towards RB19 oxidation. [47] Copyright 2016 American Chemical Society. (C) The proposed reaction upon RB19 oxidation as catalyzed by DyP.…”

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

“…The two domains, alpha and beta, form the active site cavity that hosts heme. To date, 39 crystal structures of DyPs are known (Table 1) [64,70,71,72,75,[77][78][79][80][81][82][83][84][85][86][87][88][89][90]. Detailed studies of the catalytic cycle show that hydrogen peroxide deprotonation is the first step (Fig.…”

“…Cross-linking experiments, where enzymes are incubated with hydrogen peroxide at different pH to test the possibility of forming covalent dimers by SDS-Page, allowed the identification of the specific surface residues involved. Shrestha et al reported the involvement of W263 in substrate oxidation in a class A DyP from Thermomonospora curvata [77]. Mutation of the tryptophan resulted in 50% loss of activity and sigmoidal kinetics for Reactive Blue 19 dye (RB19) as substrate.…”