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

Abstract

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (kcat(app)/Km(app) = (1.7 × 10(7)) m(-1) s(-1)) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp(220) and Arg(327) are found necessary for compound I formation, His(312) is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His(312) and Arg(327) has significant effects on the oligomerization and redox potential (E°') of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°' to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.

Keywords: dye-decolorizing peroxidase; enzyme kinetics; heme; lignin degradation; oligomerization; oxidation-reduction (redox); stopped-flow spectroscopy.

FIGURE 1.

Comparison of sequences and structures. A, sequence alignment of four types of DyPs. Putative catalytic residues surrounding the heme center are highlighted. The fingerprint motif of DyPs is boxed. B, predicted structure and active site of TcDyP using COACH based on a DyP-type peroxidase from Stretomyces coelicolor (Protein Data Bank code 4GRC). The sequence identity between the two proteins and C-score of the homology model are 42% and 0.98, respectively. C, structure and active site of HRP (Protein Data BAnk code 1ATJ).

FIGURE 2.

Biochemical characterization of TcDyP. A, SDS-PAGE of wt- and mutant TcDyP. B, UV-visible spectra of wt-TcDyP in apo (dotted line) and holo (solid line) forms. C, pyridine hemochrome assay of TcDyP calculated by the absorbance difference between the reduced (solid line) and oxidized (dotted line) spectra using Δϵ = 20.7 mm⁻¹ cm⁻¹. D, rate pH profiles of wt-TcDyP with ABTS (solid line), HQ (dash line), and guaiacol (dotted line). E, profiles of optimal assay temperature (solid line) and enzyme stability (dotted line).

FIGURE 3.

Profiles of enzyme activities. A, the wt-TcDyP with various substrates. B, activities of wt and mutant enzymes with ABTS (solid bar), HQ (line patterned bar), and guaiacol (cross-patterned bar).

FIGURE 4.

Degradation of lignin dimers by wt-TcDyP. A, structures of lignin model compounds and degradation HPLC profiles of 1 monitored at 254 nm. B, ESI-MS of the HPLC peak at 17.5 (left) and 20.8 (right) min. C, proposed pathway for the formation of degradation products.

FIGURE 5.

Degradation of MMA by wt-TcDyP. A, oxidative decarboxylation of MMA 3 to anisaldehyde 4. B, HPLC profiles monitored at 254 nm.

FIGURE 6.

Reactions of wt-TcDyP with H₂O₂. A, spectral transition of 5 μm enzyme and 5 μm H₂O₂ at pH 7.8 (left) and 3.0 (right) recorded in 2.0 s. The blue, green, and red lines represent TcDyP-0, TcDyP-I, and decay product, respectively. Arrows indicate changes of absorbance over time. B, typical time trace at 406 nm (left) and dependence of k_obs (406 nm) versus [H₂O₂] (right) for TcDyP-I formation at pH 7.8. C, typical time trace at 416 nm (left) and dependence of k_obs (416 nm) versus [H₂O₂] (right) for decay of TcDyP-I at pH 7.8. D, same as B except that the pH was 3.0.

FIGURE 7.

Reactions of TcDyP-I with HQ in a sequential mixing mode. A, spectral transition of 3 mm HQ and TcDyP-I at pH 7.8 (top) and 3.0 (bottom) recorded in 1.0 s. The green and red lines represent TcDyP-I and TcDyP-II, respectively. Arrows indicate changes of absorbance over time. B, typical time trace at 416 nm (left) and dependence of k_obs (416 nm) versus [HQ] (right) for TcDyP-II formation at pH 7.8.

FIGURE 8.

Reductions of TcDyP-II with HQ in a sequential mixing mode. A, spectral transition of 5 mm HQ and TcDyP-II at pH 3.0 recorded in 1.0 s. The blue and red lines represent TcDyP-0 and TcDyP-II, respectively. Arrows indicate changes of absorbance over time. B, typical time trace at 406 nm (left panel) and dependence of k_obs (406 nm) versus [HQ] (right panel) for TcDyP-II reduction.

FIGURE 9.

Reactions of mutants with H₂O₂ (A–D) and spectral overlay (E and F). The blue, green, and red lines in A–D represent initial, intermediate, and final states of the mutants, respectively. Reactions were performed with 5 μm mutants and different concentrations of H₂O₂ at pH 7.8 and monitored for 5 s. Arrows indicate changes of absorbance over time. The black, red, green, blue, and cyan lines in E and F represent overlay of normalized spectra of wt, D220A, H312A, H312C, and R327A, respectively. A, D220A with 5 mm H₂O₂. B, H312A with 5 μm H₂O₂. C, H312C with 5 μm H₂O₂. D, R327A with 500 μm H₂O₂. E, enzyme resting state. F, compound I.

FIGURE 10.

Formation and reduction of TcDyP-II with H312A. The reactions were performed in a sequential mixing mode. Arrows indicate changes of absorbance over time. A, spectral transition of 5 mm HQ and 5 μm TcDyP-I at pH 7.8 recorded in 1.0 s (formation of TcDyP-II). The green and red lines represent TcDyP-I and TcDyP-II, respectively. B, spectral transition of 5 mm HQ and 5 μm TcDyP-II at pH 3.0 recorded in 10 s (reduction of TcDyP-II). The blue, green, and red lines represent TcDyP-II, an intermediate state at 30 ms, and formed new species, respectively.

FIGURE 11.

Spectroelectrochemical titration of TcDyP monitored by ΔA_{433 nm}. A, spectral transition of oxidizing Fe²⁺-wt-TcDyP (blue) to Fe³⁺-wt-TcDyP (red). B, two individual titrations of wt-TcDyP at pH 6.0 (black), 7.0 (red), and 8.0 (blue). C, two individual titrations of D220A (red), H312A (blue), and R327A (pink, monitored by ΔA_{428 nm}) at pH 7.0.

FIGURE 12.

SEC profile of TcDyP. The standard curve is shown as the inset.

FIGURE 13.

Catalytic cycle of wt-TcDyP. Second-order rate constants are shown for each step.