Publications by authors named "Maria Camilla Baratto"

38 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

Chemical Profile, Antioxidant, Anti-Proliferative, Anticoagulant and Mutagenic Effects of a Hydroalcoholic Extract of Tuscan .

Plants (Basel) 2021 Jan 6;10(1). Epub 2021 Jan 6.

Department of Physical Sciences, Earth and Environment, University of Siena, Strada Laterina 8, 53100 Siena, Italy.

This study aimed to characterize the chemical profile of an ethanolic extract of Tuscan (ex) and to determine its in vitro bioactivity. The content of phenolic and flavonoid compounds, hydroxycinnamic acids and triterpenoids was determined, and high-performance liquid chromatography-diode array detection (HPLC-DAD) analysis revealed that rosmarinic acid and other hydroxycinnamic derivatives were the main constituents of the extract. ex demonstrated to have both antioxidant activity and the capability to scavenge hydrogen peroxide in a concentration dependent manner. Moreover, NIH3T3 mouse fibroblasts and human breast adenocarcinoma cells MDA-MB-231 viability was influenced by the extract with an IC of 2.4 × 10 mg/mL and 4.8 × 10 mg/mL, respectively. The addition of ex to the culture medium of both the above cell lines, resulted also in the reduction of cell death after HO pre-treatment. The Ames test demonstrated that ex was not genotoxic towards both TA98 and TA100 strains, with and without S9 metabolic activation. The extract, by inactivating thrombin, showed to also have an anti-coagulating effect at low concentration values. All these biological activities exerted by ex are tightly correlated to its phytochemical profile, rich in bioactive compounds.
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http://dx.doi.org/10.3390/plants10010097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825123PMC
January 2021

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

Binding and Reactivity of Copper to R and R Fragments of tau Protein.

Inorg Chem 2020 Jan 10;59(1):274-286. Epub 2019 Dec 10.

Dipartimento di Chimica , Università di Pavia , Via Taramelli 12 , 27100 Pavia , Italy.

Tau protein is present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble neurofibrillary tangles of tau are associated with several neurological disorders known as tauopathies, among which is Alzheimer's disease. In neurons, tau binds tubulin through its microtubule binding domain which comprises four imperfect repeats (R-R). The histidine residues contained in these fragments are potential binding sites for metal ions and are located close to the regions that drive the formation of amyloid aggregates of tau. In this study, we present a detailed characterization through potentiometric and spectroscopic methods of the binding of copper in both oxidation states to R and R peptides, which contain one and two histidine residues, respectively. We also evaluate how the redox cycling of copper bound to tau peptides can mediate oxidation that can potentially target exogenous substrates such as neuronal catecholamines. The resulting quinone oxidation products undergo oligomerization and can competitively give post-translational peptide modifications yielding catechol adducts at amino acid residues. The presence of His-His tandem in the R peptide strongly influences both the binding of copper and the reactivity of the resulting copper complex. In particular, the presence of the two adjacent histidines makes the copper(I) binding to R much stronger than in R. The copper-R complex is also much more active than the copper-R complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.
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http://dx.doi.org/10.1021/acs.inorgchem.9b02266DOI Listing
January 2020

Addition of new catalytic sites on the surface of versatile peroxidase for enhancement of LRET catalysis.

Enzyme Microb Technol 2019 Dec 12;131:109429. Epub 2019 Sep 12.

Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja California, Mexico. Electronic address:

Versatile peroxidase (VP) from Bjerkandera adusta is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants k and K were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109429DOI Listing
December 2019

Chemical characterization and antioxidant properties of products and by-products from L.

Food Sci Nutr 2019 Sep 10;7(9):2907-2920. Epub 2019 Aug 10.

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

The products and by-products of L.: olive fruits (primary agricultural product), oils (primary agro-industrial product), pomaces (agro-industrial processing by-product), and leaves (agricultural practices by-product), are promising sources of bioactive compounds. In the present study, qualitative and quantitative analyses of selected bioactive components in olive fruits, oils, and pomaces were performed. Total polyphenol content and antioxidant activity were analyzed in all samples (humid pomaces 2015: TPP, 26.0 ± 1.5-43.7 ± 3.0 g(GAEq)/kg DW; TEAC/ABTS, 189.5 ± 3.7-388.1 ± 12.0 mmol(Trx)kg DW). Radical (DPPH) quenching potential was analyzed via photometric and EPR methods, obtaining Vis/EPR signal ratio by 1.05 ± 0.45 and 1.66 ± 0.39 for fruits and pomaces, respectively. Through HPLC-UV and HPLC-MS/MS techniques, oleuropein and hydroxytyrosol, as well as selected hydroxycinnamic acids and flavonoids, were identified and quantified in olive fruits and pomaces. The main components were rutin, luteolin, and chlorogenic acid. Cytotoxic assay on fibroblast cells revealed toxic effects for selected extracts at highest tested concentrations (5%).
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http://dx.doi.org/10.1002/fsn3.1142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766567PMC
September 2019

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

Fibrils of α-Synuclein Abolish the Affinity of Cu-Binding Site to His50 and Induce Hopping of Cu Ions in the Termini.

Inorg Chem 2019 Aug 1;58(16):10920-10927. Epub 2019 Aug 1.

Department of Chemistry , Ben-Gurion University of the Negev , Be'er Sheva 84105 , Israel.

The effect of Cu on α-synuclein (AS) aggregation is important because clinical studies of patients with Parkinson's disease have shown elevated levels of Cu in the cerebrospinal fluid. So far, the molecular architectures of Cu-AS fibril complexes at atomic resolution are unknown. The current work identifies for the first time that His50 cannot bind Cu ions in mature fibrils. Moreover, it shows hopping of Cu ions between residues in AS fibrils and changes in the Cu coordination mode in Cu ions that bind in the termini of AS. The current study combines extensive experimental techniques, density functional theory calculations, and computational modeling tools to provide a complete description of the Cu binding site in AS fibrils. Our findings illustrate for the first time the specific interactions between Cu ions and AS fibrils, suggesting a new mechanistic perspective on the effect of Cu ions on AS aggregation.
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http://dx.doi.org/10.1021/acs.inorgchem.9b01337DOI Listing
August 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

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

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

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

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

Salinity stress constrains photosynthesis in Fraxinus ornus more when growing in partial shading than in full sunlight: consequences for the antioxidant defence system.

Ann Bot 2014 Sep 8;114(3):525-38. Epub 2014 Jul 8.

The National Research Council of Italy, Department of Biology, Agriculture and Food Sciences, Institute for Plant Protection, Via Madonna del Piano 10, I-50019, Sesto Fiorentino, Florence Italy

Background And Aims: A major challenge in plant ecophysiology is understanding the effects of multiple sub-optimal environmental conditions on plant performance. In most Mediterranean areas soil salinity builds up during the summer because of low availability of soil water coupled with hot temperatures. Although sunlight and soil salinity may strongly interact in determining a plant's performance, this has received relatively little attention.

Methods: Two-year-old seedlings of Fraxinus ornus were grown outdoors in pots during a Mediterranean summer in either 45 % (shaded plants) or 100 % (sun plants) sunlight irradiance and were supplied with either deionized water or deionized water plus 75 mm NaCl. Morpho-anatomical traits, water and ionic relations, gas exchange and photosystem II performance, concentrations of individual carotenoids, activity of antioxidant enzymes, concentrations of ascorbic acid and individual polyphenols were measured in leaves. Leaf oxidative stress and damage were estimated by in vivo analysis of stable free radicals and ultrastructural analyses.

Key Results: Leaf concentrations of potentially toxic ions did not markedly differ in shaded or sun plants in response to salinity. Leaves of sun plants displayed superior water use efficiency compared with leaves of shaded plants, irrespective of salinity treatment, and had both better stomatal control and higher CO2 carboxylation efficiency than leaves of shaded plants. In the salt-treated groups, the adverse effects of excess midday irradiance were greater in shade than in sun plants. The activity of enzymes responsible for detoxifying hydrogen peroxide decreased in shaded plants and increased in sun plants as a result of salinity stress. In contrast, the activity of guaiacol peroxidase and the concentration of phenylpropanoids increased steeply in response to salinity in shaded plants but were unaffected in sun plants.

Conclusions: It is concluded that salinity may constrain the performance of plants growing under partial shading more severely than that of plants growing under full sun during summer. The results suggest co-ordination within the antioxidant defence network aimed at detoxifying salt-induced generation of reactive oxygen species.
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http://dx.doi.org/10.1093/aob/mcu130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204663PMC
September 2014

Effective mutations in a high redox potential laccase from Pleurotus ostreatus.

Appl Microbiol Biotechnol 2014 Jun 26;98(11):4949-61. Epub 2014 Jan 26.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cinthia 4, 80126, Naples, Italy.

Since the first report on a laccase, there has been a notable development in the interest towards this class of enzymes, highlighted from the number of scientific papers and patents about them. At the same time, interest in exploiting laccases-mainly high redox potential-for various functions has been growing exponentially over the last 10 years. Despite decades of work, the molecular determinants of the redox potential are far to be fully understood. For this reason, interest in tuning laccase redox potential to provide more efficient catalysts has been growing since the last years. The work herein described takes advantage of the filamentous fungus Aspergillus niger as host for the heterologous production of the high redox potential laccase POXA1b from Pleurotus ostreatus and of one of its in vitro selected variants (1H6C). The system herein developed allowed to obtain a production level of 35,000 U/L (583.3 μkat/L) for POXA1b and 60,000 U/L (1,000 μkat/L) for 1H6C, corresponding to 13 and 20 mg/L for POXA1b and 1H6C, respectively. The characterised proteins exhibit very similar characteristics, with some exceptions regarding catalytic behaviour, stability and spectro-electrochemical properties. Remarkably, the 1H6C variant shows a higher redox potential with respect to POXA1b. Furthermore, the spectro-electrochemical results obtained for 1H6C make it tempting to claim that we spectro-electrochemically determined the redox potential of the 1H6C T2 site, which has not been studied in any detail by spectro-electrochemistry yet.
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http://dx.doi.org/10.1007/s00253-013-5491-8DOI 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

Prediction model based on decision tree analysis for laccase mediators.

Enzyme Microb Technol 2013 Jan 1;52(1):68-76. Epub 2012 Nov 1.

Departamento de Química Orgánica, Grupo de Química Biológica y Computacional, Universidad de Concepción, Edmundo Larenas 129, Concepción, Chile.

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.
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http://dx.doi.org/10.1016/j.enzmictec.2012.10.009DOI Listing
January 2013

A Mn(II)-Mn(II) center in human prolidase.

Biochim Biophys Acta 2013 Jan 19;1834(1):197-204. Epub 2012 Sep 19.

Department of Molecular Medicine, Section of Biochemistry, University of Pavia, via Taramelli 3 b-27100 Pavia, Italy.

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)-Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.
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http://dx.doi.org/10.1016/j.bbapap.2012.09.008DOI Listing
January 2013

Influence of pH on the speciation of copper(II) in reactions with the green tea polyphenols, epigallocatechin gallate and gallic acid.

J Inorg Biochem 2012 Jul 30;112:10-6. Epub 2011 Dec 30.

Health and Environment Department, Environmental Resources and Technologies, AIT Austrian Institute of Technology GmbH, Konrad-Lorenz-Strasse 24, 3430 Tulln, Austria.

Changes in speciation of copper(II) in reactions with epigallocatechin gallate (EGCG) and gallic acid (GA) as a function of pH have been investigated by multifrequency (X- and S-band) EPR spectroscopy in the fluid and frozen states. The EPR spectra show the formation of three distinct mononuclear species with each of the polyphenols, and these are interpreted in terms of one mono- and two bis-complexes. However, di- or polymeric complexes dominate the Cu(II) speciation in the pH range 4-8, and it is only at alkaline pH values that these mononuclear complexes make appreciable contributions to the metal speciation. Each mononuclear complex displays linewidth anisotropy in fluid solution as a consequence of incomplete averaging of the spin Hamiltonian parameters through molecular motion. Rotational correlation times for the individual complexes have been estimated by analysing the lineshape anisotropy of the fluid solution spectra using parameters determined by simulation of the rigid limit spectra. These show that the molecular masses increase with increasing pH, indicating either coordination of increasing numbers of polyphenol molecules as ligands to the copper or the increasing involvement of polyphenol dimers as ligands in the copper coordination sphere.
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http://dx.doi.org/10.1016/j.jinorgbio.2011.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401972PMC
July 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

The role of His-50 of α-synuclein in binding Cu(II): pH dependence, speciation, thermodynamics and structure.

Metallomics 2011 Mar 6;3(3):292-302. Epub 2011 Jan 6.

Dipartimento di Chimica, Università di Siena, via Aldo Moro, 53-100 Siena, Italy.

Copper interaction with alpha synuclein (αS) has been shown to accelerate aggregation and oligomerization of the protein. Three different αS copper binding domains have been proposed: (i) the N-terminal residues (1-9) that represent the minimal copper binding domain; (ii) the His-50 imidazole and (iii) the Asp and Glu residues within the acidic C-terminal domain. The copper coordination at the N-terminus has been extensively characterized and it is generally accepted that it provides the highest affinity site. The same does not hold for the role played by His-50 in copper binding. In this work Cu(ii) coordination to peptide fragments encompassing residues 45-55 of αS has been exhaustively characterized, including systems containing the inherited mutations E46K and A53T, as model peptides of the His-50 site. Through potentiometric titrations all the speciation profiles have been determined and the stability constants have been used to estimate the dissociation constants of complexes corresponding to the binding modes at pH 6.5 and 7.5. Spectroscopic analyses allowed determination of (i) the copper coordination sphere, (ii) its geometry and (iii) the constraints wherefrom the 3D structural models of the copper complexes could be obtained.
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http://dx.doi.org/10.1039/c0mt00068jDOI Listing
March 2011

Synthesis and reactivity studies on new copper(II) complexes: DNA binding, generation of phenoxyl radical, SOD and nuclease activities.

Eur J Med Chem 2010 Sep 19;45(9):3770-9. Epub 2010 May 19.

Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee-247667, Uttarakhand, India.

Tridentate ligand PhimpH binds to Cu(II) centre after deprotonation affording a new family of complexes [Cu(Phimp)(H(2)O)](2)(ClO(4))(2) (1), [Cu(Phimp)(2)] (2) and [Cu(Phimp)(L)] (3-5) where L are CH(3)COO(-), SCN(-) and NO(2)(-) respectively. The molecular structures of complexes 1 x CH(3)CN and 3 were determined by X-ray crystallography. Electrochemical studies depicted Cu(II)/Cu(I) couple in the range of -0.50 to -0.65 V vs. Ag/AgCl. EPR spectral studies for complexes 4 and 5 indicated the order of covalent character in 4 > 5 with d(x(2)-y(2)) ground state. Novel copper complexes were synthesized by a tridentate ligand having phenolato, pyridine and azomethine nitrogen donors. Phenoxyl radical complexes were generated; DNA interaction, SOD and nuclease activities were investigated. [corrected]
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http://dx.doi.org/10.1016/j.ejmech.2010.05.026DOI Listing
September 2010