Special Issue on Biotechnological Applications of Oxidoreductases.

This Special Issue was launched in conjunction with the 10th edition of the OxiZymes meeting in Siena (Italy) in 2022 [...].

A Combined NMR and UV-Vis Approach to Evaluate Radical Scavenging Activity of Rosmarinic Acid and Other Polyphenols.

Oxidative stress results from an imbalance between reactive oxygen species (ROS) production and the body's ability to neutralize them. ROS are reactive molecules generated during cellular metabolism and play a crucial role in normal physiological processes. However, excessive ROS production can lead to oxidative damage, contributing to various diseases and aging. This study is focused on rosmarinic acid (RA), a hydroxycinnamic acid (HCA) derivative well known for its antioxidant activity. In addition, RA has also demonstrated prooxidant behavior under specific conditions involving high concentrations of transition metal ions such as iron and copper, high pH, and the presence of oxygen. In this study, we aim to clarify the underlying mechanisms and factors governing the antioxidant and prooxidant activities of RA, and to compare them with other HCA derivatives. UV-Vis, NMR, and EPR techniques were used to explore copper(II)'s binding ability of RA, caffeic acid, and p-coumaric acid. At the same time, UV-Vis and NMR methods were exploited to evaluate the polyphenols' free radical scavenging abilities towards ROS generated by the ascorbic acid-copper(II) system. All the data indicate that RA is the most effective polyphenol both in copper binding abilities and ROS protection.

Quantification and Improvement of the Dynamics of Human Serum Albumin and Glycated Human Serum Albumin with Astaxanthin/Astaxanthin-Metal Ion Complexes: Physico-Chemical and Computational Approaches.

Glycated human serum albumin (gHSA) undergoes conformational changes and unfolding events caused by free radicals. The glycation process results in a reduced ability of albumin to act as an endogenous scavenger and transporter protein in diabetes mellitus type 2 (T2DM) patients. Astaxanthin (ASX) in native form and complexed with metal ions (Cu2+ and Zn2+) has been shown to prevent gHSA from experiencing unfolding events. Furthermore, it improves protein stability of gHSA and human serum albumin (HSA) as it is shown through molecular dynamics studies. In this study, the ASX/ASX-metal ion complexes were reacted with both HSA/gHSA and analyzed with electronic paramagnetic resonance (EPR) spectroscopy, rheology and zeta sizer (particle size and zeta potential) analysis, circular dichroism (CD) spectroscopy and UV-Vis spectrophotometer measurements, as well as molecular electrostatic potential (MEP) and molecular docking calculations. The addition of metal ions to ASX improves its ability to act as an antioxidant and both ASX or ASX-metal ion complexes maintain HSA and gHSA stability while performing their functions.

Lipoic/Capsaicin-Related Amides: Synthesis and Biological Characterization of New TRPV1 Agonists Endowed with Protective Properties against Oxidative Stress.

α-Lipoic acid is a sulfur-containing nutrient endowed with pleiotropic actions and a safe biological profile selected to replace the unsaturated alkyl acid of capsaicin with the aim of obtaining lipoic amides potentially active as a TRPV1 ligand and with significant antioxidant properties. Thus, nine compounds were obtained in good yields following a simple synthetic procedure and tested for their functional TRPV1 activity and radical-scavenger activity. The safe biological profile together with the protective effect against hypoxia damage as well as the in vitro antioxidant properties were also evaluated. Although less potent than capsaicin, almost all lipoic amides were found to be TRPV1 agonists and, specifically, compound 4, the lipoic analogue of capsaicin, proved to be the best ligand in terms of efficacy and potency. EPR experiments and in vitro biological assays suggested the potential protective role against oxidative stress of the tested compounds and their safe biological profile. Compounds 4, 5 and 9 significantly ameliorated the mitochondrial membrane potential caused by hypoxia condition and decreased F2-isoprostanes, known markers of oxidative stress. Thus, the experimental results encourage further investigation of the therapeutic potential of these lipoic amides.

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

Pyomelanin mimics from homogentisic acid (HGA) and gentisic acid (GA) were biosynthesized by the oxidative enzyme T. versicolor 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.

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

Belladine N-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 N-oxides were active against influenza A virus, involving the early stage of the virus replication life cycle.

Binding and Reactivity of Copper to R1 and R3 Fragments of tau Protein.

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 (R1-R4). 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 R1 and R3 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 R3 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 R3 much stronger than in R1. The copper-R3 complex is also much more active than the copper-R1 complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.

Fibrils of α-Synuclein Abolish the Affinity of Cu2+-Binding Site to His50 and Induce Hopping of Cu2+ Ions in the Termini.

The effect of Cu2+ on α-synuclein (AS) aggregation is important because clinical studies of patients with Parkinson's disease have shown elevated levels of Cu2+ in the cerebrospinal fluid. So far, the molecular architectures of Cu2+-AS fibril complexes at atomic resolution are unknown. The current work identifies for the first time that His50 cannot bind Cu2+ ions in mature fibrils. Moreover, it shows hopping of Cu2+ ions between residues in AS fibrils and changes in the Cu2+ coordination mode in Cu2+ 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 Cu2+ binding site in AS fibrils. Our findings illustrate for the first time the specific interactions between Cu2+ ions and AS fibrils, suggesting a new mechanistic perspective on the effect of Cu2+ ions on AS aggregation.

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).

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.

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

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus 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 O2-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 H2O2, 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.

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

An actinobacteria strain was isolated from Algerian Sahara soil and assigned to Streptomyces cyaneofuscatus 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 S. cyaneofuscatus 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 Trametes versicolor (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 S. cyaneofuscatus Pridham et al. 1958 represents a new biotechnological alternative to commercial insoluble pigments.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Copper Binding and Redox Activity of α-Synuclein in Membrane-Like Environment.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu+ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu+ is stabilized and is unreactive in the presence of O2 in catechol substrate oxidation.