Redox-Active Sites in <i>Auricularia auricula-judae</i> Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study

Baratto, Maria Camilla; Sinicropi, Adalgisa; Linde, Dolores; Sáez-Jiménez, Verónica; Sorace, Lorenzo; Ruíz-Dueñas, Francisco J.; Martı́nez, Ángel T.; Basosi, Riccardo; Pogni, Rebecca

doi:10.1021/acs.jpcb.5b02961

Cited by 18 publications

(34 citation statements)

References 64 publications

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“…Subtraction of Y332F from the wt gave spectral features of g x , g y , and g z values at 2.0078, 2.0049, and 2.0025 (the dotted line in Figure 3D), respectively, which is consistent with the previous observed g values for tyrosyl radicals. 50 The broad peaks in Y332F, showing g values ranging from 2.002 to 2.005, were likely resulted from tryptophanyl and other unknown radicals.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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“…In recent years, dye-decolorizing peroxidases (DyPs) have also been shown to harbor amino acid radical sites. 6 , 8 , 24 , 25 DyPs have attracted interest due to their activity toward anthraquinone dyes and phenolic lignin model compounds. 26 The DyP family is composed of three phylogenetically, structurally, and biochemically distinct subfamilies (types A, B, and C/D).…”

mentioning

confidence: 99%

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy

et al. 2021

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The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase ( Kp DyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of Kp DyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in Kp DyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

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“…2B). This was also confirmed by the Trp •+ radical signal in electron paramagnetic resonance (EPR) spectra, which was not observed for W377S AajDyP mutant [48,49].…”

Section: Distribution Of Electron Spin By Qm/mm Calculations On Tyr37mentioning

confidence: 77%

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

Lin

2019

Biotech and App Biochem

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Environmental pollutants, such as industrial dyes and halophenols, are harmful to human health, which urgently demand degradation. Bioremediation has been shown to be a cost‐effective and ecofriendly approach. As reviewed herein, significant progress has been made in the last decade for biodegradation of both industrial dyes and halophenols, by engineering of native dye‐decolorizing peroxidases (DyPs) and dehaloperoxidases (DHPs), and by design of artificial heme enzymes in both native and de novo protein scaffolds. The catalytic efficiency of artificial DyPs and DHPs can be rationally designed comparable to or even beyond those of natural counterparts. The enzymes are on their way from laboratory to industry and will play more crucial roles in environmental protection toward a green future.

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Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study

Cited by 18 publications

References 64 publications

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

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

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy

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

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