Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

Silveira, Célia M.; Moe, Elin; Fraaije, Marco W.; Martins, Lı́gia O.; Todorović, Smilja

doi:10.1039/d0ra00950d

Cited by 6 publications

(15 citation statements)

References 46 publications

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“…These RR band frequencies suggest the presence of a strong sixth ligand (six coordinated LS, 6cLS state) in the resting ferric AncDyPD-b1, most likely OH À , H 2 O, or some other small molecule (vide infra). This finding is consistent with the lack of catalytic activity of the enzyme at neutral pH, since the presence of the sixth axial ligand prevents H 2 O 2 from binding to the active site, as already observed in some bacterial DyPs [39]. RR spectrum obtained at pH 3.5 reveals the presence of another species, indicated by downshifted components of m 3 and m 2 bands at 1488 and 1573 cm À1 , respectively, which can be attributed to a five coordinated high spin (5cHS) population.…”

Section: Expression and Purificationsupporting

confidence: 90%

“…The catalytically competent 5cHS population [39,40] represents around 35 % in AncDyPD-b1 at pH 3.5, as judged by the comparison of the intensities of the two m 3 bands, which is consistent with the observed acidic optimum pH for enzyme activity. Interestingly, highly similar pH dependence of RR spectra has been recently reported for a C-type DyP from Deinococcus radiodurans, DrDyP [39]. We can hence correlate the observed Soret band at 426 nm of AncDyPD-b1 with an inactive 6cLS species, while the active 5cHS population is most likely characterized by the observed 410 nm Soret band.…”

Section: Expression and Purificationsupporting

confidence: 76%

“…RR spectrum obtained at pH 3.5 reveals the presence of another species, indicated by downshifted components of m 3 and m 2 bands at 1488 and 1573 cm À1 , respectively, which can be attributed to a five coordinated high spin (5cHS) population. The catalytically competent 5cHS population [39,40] represents around 35 % in AncDyPD-b1 at pH 3.5, as judged by the comparison of the intensities of the two m 3 bands, which is consistent with the observed acidic optimum pH for enzyme activity. Interestingly, highly similar pH dependence of RR spectra has been recently reported for a C-type DyP from Deinococcus radiodurans, DrDyP [39].…”

Section: Expression and Purificationsupporting

confidence: 76%

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Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

et al. 2021

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Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process.

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Section: Expression and Purificationsupporting

confidence: 90%

Section: Expression and Purificationsupporting

confidence: 76%

Section: Expression and Purificationsupporting

confidence: 76%

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Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

et al. 2021

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“…Furthermore, DyPs lack the highly conserved catalytic distal His residue. In turn, the distal side of the heme pocket typically contains an Asp (or Glu) and an Arg, which are thought to be involved in the catalytic reaction, although their exact roles are not well understood yet [145][146][147]149].…”

Section: Dye-decolorizing Peroxidasesmentioning

confidence: 99%

Electrocatalysis by Heme Enzymes—Applications in Biosensing

et al. 2021

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Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.

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“…In order to provide a reference for the studies of immobilised enzymes, the RR spectra of as purified, ferric DyPs were measured in solution. The high frequency region of the RR spectra of CboDyP show oxidation (ν4) and oxidation/spin state (ν3 and ν2) marker bands at 1371, 1481 and 1561 cm −1 at pH 8, which are indicative of a uniform and homogeneous 6-coordinated high-spin (6cHS) population (Figure 2B) [26]. DyPs in solution.…”

Section: Evaluation Of Structural Integrity Of Dyps Upon Immobilisationmentioning

confidence: 99%

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

Zuccarello

Barbosa

Galdino

et al. 2021

IJMS

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Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

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Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

Abstract: The solution active site architecture of DyPs, which is sensitively revealed by RR spectroscopy, is crucial for comprehension of their physiological roles and for evaluation of their potential for biotechnological application.

Cited by 6 publications

References 46 publications

Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

Electrocatalysis by Heme Enzymes—Applications in Biosensing

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

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