Publications by authors named "Rebecca Pogni"

54 Publications

Stereoselective Access to Antimelanoma Agents by Hybridization and Dimerization of Dihydroartemisinin and Artesunic acid.

ChemMedChem 2021 Apr 1. Epub 2021 Apr 1.

Department of Biological and Ecological Sciences, Univeristy of Viterbo, Via S.C. De Lellis s.n.c., 01100, Viterbo, Italy.

A library of five hybrids and six dimers of dihydroartemisinin and artesunic acid has been synthetized in a stereo-controlled manner and evaluated for the anticancer activity against metastatic melanoma cell line (RPMI7951). Among novel derivatives, three artesunic acid dimers showed antimelanoma activity and cancer selectivity, being not toxic on normal human fibroblast (C3PV) cell line. Among the three dimers, the one bearing 4-hydroxybenzyl alcohol as a spacer showed no cytotoxic effect (CC >300 μM) and high antimelanoma activity (IC =0.05 μM), which was two orders of magnitude higher than that of parent artesunic acid, and of the same order of commercial drug paclitaxel. In addition, this dimer showed cancer-type selectivity towards melanoma compared to prostate (PC3) and breast (MDA-MB-231) tumors. The occurrence of a radical mechanism was hypothesized by DFO and EPR analyses. Qualitative structure activity relationships highlighted the role of artesunic acid scaffold in the control of toxicity and antimelanoma activity.
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http://dx.doi.org/10.1002/cmdc.202100196DOI Listing
April 2021

Laccase-Catalyzed 1,4-Dioxane-Mediated Synthesis of Belladine -Oxides with Anti-Influenza A Virus Activity.

Int J Mol Sci 2021 Jan 29;22(3). Epub 2021 Jan 29.

Department of Ecology and Biology, University of Tuscia, 01100 Viterbo, Italy.

Belladine -oxides active against influenza A virus have been synthetized by a novel laccase-catalyzed 1,4-dioxane-mediated oxidation of aromatic and side-chain modified belladine derivatives. Electron paramagnetic resonance (EPR) analysis confirmed the role of 1,4-dioxane as a co-oxidant. The reaction was chemo-selective, showing a high functional-group compatibility. The novel belladine -oxides were active against influenza A virus, involving the early stage of the virus replication life cycle.
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http://dx.doi.org/10.3390/ijms22031337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866262PMC
January 2021

Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity.

Int J Mol Sci 2021 Feb 9;22(4). Epub 2021 Feb 9.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, Italy.

Pyomelanin mimics from homogentisic acid (HGA) and gentisic acid (GA) were biosynthesized by the oxidative enzyme laccase at physiological pH to obtain water soluble melanins. The pigments show brown-black color, broad band visible light absorption, a persistent paramagnetism and high antioxidant activity. The EPR approach shows that at least two different radical species are present in both cases, contributing to the paramagnetism of the samples. This achievement can also shed light on the composition of the ochronotic pigment in the Alkaptonuria disease. On the other hand, these soluble pyomelanin mimics, sharing physico-chemical properties with eumelanin, can represent a suitable alternative to replace the insoluble melanin pigment in biotechnological applications.
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http://dx.doi.org/10.3390/ijms22041739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916096PMC
February 2021

Structure and Bioactive Properties of Novel Textile Dyes Synthesised by Fungal Laccase.

Int J Mol Sci 2020 Mar 17;21(6). Epub 2020 Mar 17.

Department of Biochemistry and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.

Novel sustainable processes involving oxidative enzymatic catalysts are considered as an alternative for classical organic chemistry. The unique physicochemical and bioactive properties of novel bio-products can be obtained using fungal laccase as catalyst. Among them are textile biodyes synthesised during oxidation of substrates belonging to the amine and methoxy organic derivatives. The process of synthesis occurs in mild conditions of pH, temperature, and pressure, and without using harmful oxidants. The effect of fungal laccase activity on the substrates mixture transformation efficiency was analysed in terms of antimicrobial dye synthesis on a large scale. Three new phenazine dyes, obtained in the presence of laccase from , were studied for their structure and properties. The phenazine core structure of the products was a result of tri-molecular transformation of aminomethoxybenzoic acid and aminonaphthalene sulfonic acid isomers. One of the compounds from the synthesised dye, namely 10-((2-carboxy-6-methoxyphenyl)amino)-11-methoxybenzo[a]phenazine-8-carboxylic acid, was able to inhibit the growth of . The high concentration of substrates (5 g/L) was efficiently transformed during 72 h in the mild conditions of pH 4 with the use of laccase with an activity of 200 U per g of the substrates mixture. The new bioactive dye exhibited excellent dyeing properties with concomitant antibacterial and antioxidative activity. The proposed enzyme-mediated synthesis represents an alternative eco-friendly route for the synthesis of novel antimicrobial compounds with high importance for the medical textile industry.
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http://dx.doi.org/10.3390/ijms21062052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139866PMC
March 2020

Paramagnetism and Relaxation Dynamics in Melanin Biomaterials.

J Phys Chem B 2020 03 10;124(11):2110-2115. Epub 2020 Mar 10.

Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy.

Spectroscopical characterization of melanins is a prior requirement for the efficient tailoring of their radical scavenging, ultraviolet-visible radiation absorption, metal chelation, and natural pigment properties. Electron paramagnetic resonance (EPR), exploiting the common persistent paramagnetism of melanins, represents the elective standard for the structural and dynamical characterization of their constituting radical species. Although melanins are mainly investigated using X-band (9.5 GHz) continuous wave (CW)-EPR, an integration with the application of Q-band (34 GHz) in CW and pulse EPR for the discrimination of melanin pigments of different compositions is presented here. The longitudinal relaxation times measured highlight faster relaxation rates for cysteinyldopa melanin, compared to those of the most common dopa melanin pigment, suggesting pulse EPR spin-lattice relaxation time measurements as a complementary tool for characterization of pigments of interest for biomimetic materials engineering.
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http://dx.doi.org/10.1021/acs.jpcb.9b11785DOI Listing
March 2020

Synthesis and Evaluation of Artemisinin-Based Hybrid and Dimer Derivatives as Antimelanoma Agents.

ACS Omega 2020 Jan 27;5(1):243-251. Epub 2019 Dec 27.

Department of Ecological and Biological Sciences, University of Tuscia, via S. C. De Lellis 44, 01100, Viterbo, Italy.

A library of hybrid and dimer compounds based on the natural scaffold of artemisinin was synthesized. These derivatives were obtained by coupling of artemisinin derivatives, artesunate, and dihydroartemisinin with a panel of phytochemical compounds. The novel artemisinin-based hybrids and dimers were evaluated for their anticancer activity on a cervical cancer cell line (HeLa) and on three complementary metastatic melanoma cancer cell lines (SK-MEL3, SK-MEL24, and RPMI-7951). Two hybrid compounds obtained by coupling of artesunate with eugenol and tyrosol, and one of the dimer compounds containing curcumin, emerged as the most active and cancer-selective derivatives.
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http://dx.doi.org/10.1021/acsomega.9b02600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964273PMC
January 2020

Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotus ostreatus strain.

Enzyme Microb Technol 2020 Jan 17;132:109398. Epub 2019 Aug 17.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, Italy.

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of type and pH value of buffer, reaction temperature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40 ℃ and 60 ℃ in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109398DOI Listing
January 2020

Spectroscopic Characterisation of the Naphthalene Dioxygenase from sp. Strain NCIMB12038.

Int J Mol Sci 2019 Jul 11;20(14). Epub 2019 Jul 11.

Department of Biotechnology, Chemistry and Pharmacy, Via A. Moro 2, 53100 Siena, Italy.

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of HO, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.
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http://dx.doi.org/10.3390/ijms20143402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678576PMC
July 2019

Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: Synthesis, structure-activity relationships and studies on their biomimetic reaction with Fe(II).

Bioorg Chem 2019 08 31;89:103020. Epub 2019 May 31.

Department of Biotechnology, Chemistry and Pharmacy (DoE 2018-2022), University of Siena, via Aldo Moro 2, 53100 Siena, Italy.

Despite recent advancements in its control, malaria is still a deadly parasitic disease killing millions of people each year. Progresses in combating the infection have been made by using the so-called artemisinin combination therapies (ACTs). Natural and synthetic peroxides are an important class of antimalarials. Here we describe a new series of peroxides synthesized through a new elaboration of the scaffold of bicyclic-fused/bridged synthetic endoperoxides previously developed by us. These peroxides are produced by a straightforward synthetic protocol and are characterized by submicromolar potency when tested against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. To investigate their mode of action, the biomimetic reaction of the representative compound 6w with Fe(II) was studied by EPR and the reaction products were characterized by NMR. Rationalization of the observed structure-activity relationship studies was performed by molecular docking. Taken together, our data robustly support the hypothesized mode of activation of peroxides 6a-cc and led to the definition of the key structural requirements responsible for the antiplasmodial potency. These data will pave the way in future to the rational design of novel optimized antimalarials suitable for in vivo investigation.
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http://dx.doi.org/10.1016/j.bioorg.2019.103020DOI Listing
August 2019

Effect of Electrode Shape and Flow Conditions on the Electrochemical Detection with Band Microelectrodes.

Sensors (Basel) 2018 Sep 21;18(10). Epub 2018 Sep 21.

Institute for the Chemistry of Organometallic Compounds, Italian National Council for Research, 50019 Florence, Italy.

In this work, we report the analysis of the electrochemical detection of electroactive species with band microelectrodes that operate under controlled convection. The study focuses on the determination of the collection efficiency of the analyte as a function of inlet flow velocity and microband geometry (inlaid, bumped and recessed), also providing a straightforward method for the theoretical determination of the lower detection limit. The analysis has been carried out by simulating the dimensionless mass transport with the finite element method, delivering the stationary limiting current density. Simulations have been performed on systems consisting of single and double band electrodes to investigate the trail effect on the electrochemical detection. We show that the obtained dimensionless results can be easily turned into dimensional data, providing a tool for the design of devices. The proposed method is general and can easily be extended to systems with different geometry.
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http://dx.doi.org/10.3390/s18103196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210975PMC
September 2018

Spectroscopic Characterization of Natural Melanin from a Strain and Comparison with Melanin Enzymatically Synthesized by Tyrosinase and Laccase.

Molecules 2018 Aug 1;23(8). Epub 2018 Aug 1.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.

An actinobacteria strain was isolated from Algerian Sahara soil and assigned to Pridham et al. 1958 species. This strain was selected for its ability to produce melanin exopigments in liquid and solid media. Melanin synthesis was associated with tyrosinase activity and the enzyme from this strain was isolated and biochemically characterized. Synthetic melanin was then enzymatically produced using the Pridham et al. 1958 tyrosinase. As this enzyme showed a higher diphenolase activity, a synthetic melanin from the enzymic oxidation of 3,4-dihydroxyphenylalanine (dopa) was obtained by the use of a (L.) Lloyd laccase for comparison. The natural and synthetic pigments were physico-chemically characterized by the use of ultraviolet (UV)-Visible, and Fourier transform infrared (FT-IR) and multifrequency electron paramagnetic resonance (EPR) spectroscopies. All the melanin samples displayed a stable free radical when analyzed by X-band EPR spectroscopy. Once the samples were recorded at Q-band EPR, a copolymer derived from a mixture of different constituents was evident in the natural melanin. All radical species were analyzed and discussed. The use of water-soluble melanin naturally produced by Pridham et al. 1958 represents a new biotechnological alternative to commercial insoluble pigments.
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http://dx.doi.org/10.3390/molecules23081916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222888PMC
August 2018

Isolation and characterization of a novel tyrosinase produced by Sahara soil actinobacteria and immobilization on nylon nanofiber membranes.

J Biotechnol 2018 Jan 11;265:54-64. Epub 2017 Nov 11.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy. Electronic address:

In the present study different actinomycete strains were collected and isolated from Algerian Sahara soil with the aim to select novel enzymes with promising features for biotechnological applications. The Ms1 strain was selected, amongst the others, for its capability to produce melanin in different solid media. Ms1 chromosomal DNA was sequenced and the strain assigned to Streptomyces cyaneofuscatus sp. A tyrosinase (MW∼30kD) encoding sequence was identified and the corresponding enzyme was isolated and biochemically characterized. The tyrosinase showed the highest activity and stability at neutral and alkaline pH and it was able to oxidize l-DOPA at T=55°C and pH 7. The enzyme showed variable stability in presence of various water-miscible organic solvents, while it was inactivated by reducing agents. The tyrosinase activity was unaffected by NaCl and enhanced by different cations. Furthermore, the enzyme showed a higher specificity for diphenols than monophenols showing a higher diphenolase than monophenolase activity. Finally, tyrosinase was stabilized by immobilization on nylon nanofiber membranes with a payload of 82% when 1% glutaraldeyde was used. Taken all together, these results show that the enzyme displays interesting properties for biotechnological purposes.
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http://dx.doi.org/10.1016/j.jbiotec.2017.11.004DOI Listing
January 2018

Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.

Biochemistry 2017 04 20;56(13):1887-1898. Epub 2017 Mar 20.

Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.
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http://dx.doi.org/10.1021/acs.biochem.6b01281DOI Listing
April 2017

Characterisation and Antioxidant Activity of Crude Extract and Polyphenolic Rich Fractions from C. incanus Leaves.

Int J Mol Sci 2016 Aug 17;17(8). Epub 2016 Aug 17.

Department of Agrifood Production and Environmental Sciences (DiSPAA), University of Florence, 50019 Sesto Fiorentino (Florence), Italy.

Cistus incanus (Cistaceae) is a Mediterranean evergreen shrub. Cistus incanus herbal teas have been used as a general remedy in traditional medicine since ancient times. Recent studies on the antioxidant properties of its aqueous extracts have indicated polyphenols to be the most active compounds. However, a whole chemical characterisation of polyphenolic compounds in leaves of Cistus incanus (C. incanus) is still lacking. Moreover, limited data is available on the contribution of different polyphenolic compounds towards the total antioxidant capacity of its extracts. The purpose of this study was to characterise the major polyphenolic compounds present in a crude ethanolic leaf extract (CEE) of C. incanus and develop a method for their fractionation. Superoxide anion, hydroxyl and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assays were also performed to evaluate the antioxidant properties of the obtained fractions. Three different polyphenolic enriched extracts, namely EAC (Ethyl Acetate Fraction), AF1 and AF2 (Aqueos Fractions), were obtained from CEE. Our results indicated that the EAC, enriched in flavonols, exhibited a higher antiradical activity compared to the tannin enriched fractions (AF1 and AF2). These findings provide new perspectives for the use of the EAC as a source of antioxidant compounds with potential uses in pharmaceutical preparations.
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http://dx.doi.org/10.3390/ijms17081344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000740PMC
August 2016

A Spectroscopic Study of the Aggregation State of the Human Antimicrobial Peptide LL-37 in Bacterial versus Host Cell Model Membranes.

Biochemistry 2015 Nov 4;54(45):6760-8. Epub 2015 Nov 4.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena , 53100 Siena, Italy.

The LL-37 antimicrobial peptide is the only cathelicidin peptide found in humans that has antimicrobial and immunomodulatory properties. Because it exerts also chemotactic and angiogenetic activity, LL-37 is involved in promoting wound healing, reducing inflammation, and strengthening the host immune response. The key to the effectiveness of antimicrobial peptides (AMPs) lies in the different compositions of bacterial versus host cell membranes. In this context, antimicrobial peptide LL-37 and two variants were studied in the presence of model membranes with different lipid compositions and charges. The investigation was performed using an experimental strategy that combines the site-directed spin labeling-electron paramagnetic resonance technique with circular dichroism and fluorescence emission spectroscopies. LL-37 interacts with negatively charged membranes forming a stable aggregate, which can likely produce toroidal pores until the amount of bound peptide exceeds a critical concentration. At the same time, we have clearly detected an aggregate with a higher oligomeric degree for interaction of LL-37 with neutral membranes. These data confirm the absence of cell selectivity of the peptide and a more complex role in stimulating host cells.
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http://dx.doi.org/10.1021/acs.biochem.5b00813DOI Listing
November 2015

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY.

J Biol Chem 2015 Sep 3;290(38):23201-13. Epub 2015 Aug 3.

From the Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain,

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.
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http://dx.doi.org/10.1074/jbc.M115.665919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645588PMC
September 2015

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

J Phys Chem B 2015 Oct 9;119(43):13583-92. Epub 2015 Jul 9.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.

Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253-262); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction.
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http://dx.doi.org/10.1021/acs.jpcb.5b02961DOI Listing
October 2015

Basidiomycete DyPs: Genomic diversity, structural-functional aspects, reaction mechanism and environmental significance.

Arch Biochem Biophys 2015 May 28;574:66-74. Epub 2015 Jan 28.

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain. Electronic address:

The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substrates.
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http://dx.doi.org/10.1016/j.abb.2015.01.018DOI Listing
May 2015

Spectroscopic and computational characterization of laccases and their substrate radical intermediates.

Cell Mol Life Sci 2015 Mar 17;72(5):885-96. Epub 2015 Jan 17.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A Moro 2, 53100, Siena, Italy.

Laccases are multicopper oxidases which oxidize a wide variety of aromatic compounds with the concomitant reduction of oxygen to water as by-product. Due to their high stability and biochemical versatility, laccases are key enzymes to be used as eco-friendly biocatalyst in biotechnological applications. The presence of copper paramagnetic species in the catalytic site paired with the substrate radical species produced in the catalytic cycle makes laccases particularly attractive to be studied by spectroscopic approaches. In this review, the potentiality of a combined multifrequency electron paramagnetic spectroscopy /computational approach to gain information on the nature of the catalytic site and radical species is presented. The knowledge at molecular level of the enzyme oxidative process can be of great help to model new enzymes with increased efficiency and robustness.
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http://dx.doi.org/10.1007/s00018-014-1825-7DOI Listing
March 2015

EPR and LC-MS studies on the mechanism of industrial dye decolorization by versatile peroxidase from Bjerkandera adusta.

Environ Sci Pollut Res Int 2015 Jun 9;22(11):8683-92. Epub 2015 Jan 9.

Department of Biotechnology, Chemistry, and Pharmacy, University of Siena, Siena, Italy.

The mechanisms of industrial dye transformation by versatile peroxidase were elucidated. Purified versatile peroxidase from Bjerkandera adusta was able to decolorize different classes of dyes including azo and phthalocyanines, but unable to transform any of the anthraquinones tested. Kinetic constants for selected dyes were determined and the transformation products were analyzed by EPR spectroscopy and mass spectrometry. The EPR and MS analyses of the enzymatic decolorization products showed the cleavage of the azo bond in azo dyes and the total disruption of the phthalocyaninic ring in phthalocyanine dyes. The EPR analysis on two copper-containing dyes, reactive violet 5 (azo) and reactive blue 72 (phthalocyanine), showed that the transformation can or not break the metal-ion coordination bond according the dye nature. The role of the catalytic Trp172 in the dye transformation by a long-range electron transfer pathway was confirmed and the oxidation mechanisms are proposed and discussed.
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http://dx.doi.org/10.1007/s11356-014-4051-9DOI Listing
June 2015

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

Biochem J 2015 Mar;466(2):253-62

*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.

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 (k(cat) > 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 k(cat) ~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.
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http://dx.doi.org/10.1042/BJ20141211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357238PMC
March 2015

Human neutrophil peptide 1 variants bearing arginine modified cationic side chains: effects on membrane partitioning.

Biophys Chem 2014 Jun 26;190-191:32-40. Epub 2014 Apr 26.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy. Electronic address:

α-Defensins (e.g. human neutrophil peptides, HNPs) have a broad spectrum bactericidal activity contributing to human innate immunity. The positive charge of amino acid side chains is responsible for the first interaction of cationic antimicrobial peptides with negatively charged bacterial membranes. α-Defensins contain a high content of Arg residues compared to Lys. In this paper, different peptide analogs including substitution of Arg-14 respectively with N(G)-N(G')-asymmetric dimethyl-l-arginine (ADMA), N(G)-N(G')-symmetric dimethyl-l-arginine (SDMA) and Lys (R14K and R15KR14KR15K) variants have been studied to test the role of Arg guanidino group and the localized cationic charge of Lys for interaction with lipid membranes. Our findings show that all the variants have a decreased disruptive activity against the bilayer. The methylated analogs show a reduction in membrane partitioning due to the lack of their ability to form hydrogen bonds. Comparison with the native HNP-1 peptide has been discussed.
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http://dx.doi.org/10.1016/j.bpc.2014.04.003DOI Listing
June 2014

Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism.

Biochem J 2013 Jun;452(3):575-84

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.
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http://dx.doi.org/10.1042/BJ20130251DOI Listing
June 2013

Effects of the protein environment on the spectral properties of tryptophan radicals in Pseudomonas aeruginosa azurin.

J Am Chem Soc 2013 Mar 13;135(12):4822-33. Epub 2013 Mar 13.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. Moro 2, 53100 Siena, Italy.

Many biological electron-transfer reactions involve short-lived tryptophan radicals as key reactive intermediates. While these species are difficult to investigate, the recent photogeneration of a long-lived neutral tryptophan radical in two Pseudomonas aeruginosa azurin mutants (Az48W and ReAz108W) made it possible to characterize the electronic, vibrational, and magnetic properties of such species and their sensitivity to the molecular environment. Indeed, in Az48W the radical is embedded in the hydrophobic core while, in ReAz108W it is solvent-exposed. Here we use density functional theory and multiconfigurational perturbation theory to construct quantum-mechanics/molecular-mechanics models of Az48W(•) and ReAz108W(•) capable of reproducing specific features of their observed UV-vis, resonance Raman, and electron paramagnetic resonance spectra. The results show that the models can correctly replicate the spectral changes imposed by the two contrasting hydrophobic and hydrophilic environments. Most importantly, the same models can be employed to disentangle the molecular-level interactions responsible for such changes. It is found that the control of the hydrogen bonding between the tryptophan radical and a single specific surface water molecule in ReAz108W(•) represents an effective means of spectral modulation. Similarly, a specific electrostatic interaction between the radical moiety and a Val residue is found to control the Az48W(•) excitation energy. These modulations appear to be mediated by the increase in nitrogen negative charge (and consequent increase in hydrogen bonding) of the spectroscopic D2 state with respect to the D0 state of the chromophore. Finally, the same protein models are used to predict the relaxed Az48W(•) and ReAz108W(•) D2 structures, showing that the effect of the environment on the corresponding fluorescence maxima must parallel that of D0 absorption spectra.
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http://dx.doi.org/10.1021/ja400464nDOI Listing
March 2013

The defensin-lipid interaction: insights on the binding states of the human antimicrobial peptide HNP-1 to model bacterial membranes.

Biochim Biophys Acta 2013 Feb 15;1828(2):758-64. Epub 2012 Nov 15.

Department of Chemistry, University of Siena, 53100 Siena, Italy.

Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide-membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.
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http://dx.doi.org/10.1016/j.bbamem.2012.11.011DOI Listing
February 2013

The nature of tryptophan radicals involved in the long-range electron transfer of lignin peroxidase and lignin peroxidase-like systems: Insights from quantum mechanical/molecular mechanics simulations.

Proteins 2012 May 2;80(5):1476-83. Epub 2012 Mar 2.

Department of Chemistry, University of Siena, Siena 53100, Italy.

A catalytically active tryptophan radical has been demonstrated to be involved in the long-range electron transfer to the heme cofactor of lignin peroxidase (LiP) from Phanerochaete chrysosporium although no direct detection by EPR spectroscopy of the tryptophan radical intermediate has been reported to date. An engineering-based approach has been used to manipulate the microenvironment of the redox-active tryptophan site in LiP and Coprinus cinereus Peroxidase (CiP), allowing the direct evidence of the tryptophan radical species. In light of the newly available EPR experimental data, we performed a quantum mechanical/molecular mechanics computational study to characterize the tryptophan radicals in the above protein matrices as well as in pristine LiP. The nature of the tryptophan radicals is discussed together with the analysis of their environment with the aim of understanding the different behavior of pristine LiP in comparison with that of LiP and CiP variants.
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http://dx.doi.org/10.1002/prot.24046DOI Listing
May 2012

Insights into the homocoupling reaction of 4-methylamino benzoic acid mediated by Trametes versicolor laccase.

Mol Biosyst 2011 Nov 12;7(11):2967-9. Epub 2011 Sep 12.

Department of Chemistry, via A. De Gasperi 2, 53100, Siena, Italy.

Spectroscopic measurements combined with Density Functional Theory calculations were applied to the characterization of the homocoupling reaction of 4-methylamino benzoic acid mediated by laccase.
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http://dx.doi.org/10.1039/c1mb05301aDOI Listing
November 2011

Structural and Functional Consequences Induced by Post-Translational Modifications in α-Defensins.

Int J Pept 2011 28;2011:594723. Epub 2011 Aug 28.

School of Biosciences and Biotechnologies, University of Camerino, Gentile III da Varano Street, 62032 Camerino, Italy.

HNP-1 is an antimicrobial peptide that undergoes proteolytic cleavage to become a mature peptide. This process represents the mechanism commonly used by the cells to obtain a fully active antimicrobial peptide. In addition, it has been recently described that HNP-1 is recognized as substrate by the arginine-specific ADP-ribosyltransferase-1. Arginine-specific mono-ADP-ribosylation is an enzyme-catalyzed post-translational modification in which NAD(+) serves as donor of the ADP-ribose moiety, which is transferred to the guanidino group of arginines in target proteins. While the arginine carries one positive charge, the ADP-ribose is negatively charged at the phosphate moieties at physiological pH. Therefore, the attachment of one or more ADP-ribose units results in a marked change of cationicity. ADP-ribosylation of HNP-1 drastically reduces its cytotoxic and antibacterial activities. While the chemotactic activity of HNP-1 remains unaltered, its ability to induce interleukin-8 production is enhanced. The arginine 14 of HNP-1 modified by the ADP-ribose is in some cases processed into ornithine, perhaps representing a different modality in the regulation of HNP-1 activities.
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http://dx.doi.org/10.1155/2011/594723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163396PMC
November 2011

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine.

J Biol Chem 2011 Apr 2;286(17):15525-34. Epub 2011 Mar 2.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain.

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.
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http://dx.doi.org/10.1074/jbc.M111.220996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083212PMC
April 2011