Over recent decades, various enzymes capable of breaking down polyethylene terephthalate (PET) have emerged as sustainable tools for plastic waste management. Among them, IsPETase from Ideonella sakaiensis 201-f6 stands out for its high catalytic activity at low temperatures. However, the discovery of the PETase-like enzyme from the marine sponge Streptomyces sp. SM14 (PETaseSM14) has introduced a new class of biocatalysts active at high-salt concentrations, whose structural and catalytic properties remain poorly understood. This study explores the structural and catalytic behavior of both IsPETase and PETaseSM14 under varying ionic strength (from 150 to 900 mM of NaCl concentration) using all-atom molecular dynamics simulations and in vitro assays. Results reveal that the flexible, enlarged binding site of IsPETase improves substrate accommodation but also causes catalytic residue displacement and rapid deactivation, particularly under high-salt conditions. In contrast, PETaseSM14 has a smaller, more rigid binding pocket that undergoes moderate widening upon salt concentration increasing, thus promoting water and substrate recruitment. Additionally, active forms of both enzymes bind PET chains in conformations similar to those found in amorphous PET. These findings offer key structural insights that can inform future enzyme engineering efforts for effective PET degradation tailored to diverse environmental conditions.

The recent discovery of the PETase enzyme family offers a sustainable solution for depolymerizing poly-(ethylene terephthalate) (PET), one of the most widespread plastic compounds, under mild conditions. This enables the environmentally beneficial conversion of plastic waste into value-added products. Among this enzyme family, PETase from Ideonella sakaiensis has been the most extensively studied. Although other similar enzymes have been discovered, our knowledge about the catalytic and structural properties of this class remains limited. In this study, a PETase-like enzyme (PETase SM14) from Streptomyces sp. SM14 was heterologously produced in Escherichia coli, and its activity was tested on post-consumer plastic substrates using high-performance liquid chromatography for product quantification as well as scanning electron microscopy and atomic force microscopy for substrate surface imaging evaluation. PETase SM14 exhibited high salt tolerance (1.5 M), good heat resistance (Tm 56.26 °C), and optimal activity at pH 9.0, highlighting its potential for PET waste bioremediation. Furthermore, its X-ray crystal structure was solved at 1.43 Å resolution, revealing conserved features of the PETase family with potential relevance for future engineering applications.

The electronic absorption, MCD, and RR spectra of the Co(III) and Co(II) derivatives of wild-type human neuroglobin (Co-WT) and its C46A/C55A mutant (Co-C46AC55A) were thoroughly investigated and compared with those of the corresponding Fe species and of the few Co-substituted heme proteins characterized so far. In both oxidation states, Co-WT and Co-C46AC55A contain a low-spin six-coordinated Co ion, whose axial coordination positions appear to be occupied by the distal and proximal histidines and whose electronic properties are scarcely affected by deletion of the C46-C55 disulfide bond. Both Co-WT and Co-C46AC55A feature negative E°'Co(III)/Co(II) values. Fe(III) to Co(III) swapping does not significantly alter the pH dependence of their spectroscopic properties and E°' values, indicating that no major changes occur in their regulating molecular factors. Most importantly, Co-WT and Co-C46AC55A can catalyze the reduction of H3O+ to H2, with onset potentials and overpotentials comparable to those of Co-porphyrin/polypeptide catalysts. The electrocatalytic efficiency of Co-WT and Co-C46AC55A for the development of H2 is slightly lower compared to that of six-coordinated aquo-His Co-Mb, although they are less affected by the presence of dioxygen.

Laccases that oxidize low-density polyethylene (LDPE) represent a promising strategy for bioremediation purposes. To rationalize or optimize their PE-oxidative activity, two fundamental factors must be considered: the enzyme‘s redox potential and its binding affinity/mode towards LDPE. Indeed, a stable laccase-PE complex may facilitate a thermodynamically unfavorable electron transfer, even without redox mediators. In this study, we compared the redox potential and the LDPE-binding properties of three different PE-oxidizing laccases: a fungal high-redox potential laccase from Trametes versicolor , a bacterial low-redox potential laccase from Bacillus subtilis , and the recently characterized LMCO2 from Rhodococcus opacus R7. First we found that LMCO2 is a low-potential laccase (E°=413 mV), as reported in other bacterial variants. Using computational tools, we simulated the interactions of these laccases with a large LDPE model and highlighted the key role of hydrophobic residues surrounding the T1 site. Notably, a methionine-rich loop in LMCO2 appears to enhance the formation of a stable complex with LDPE, potentially facilitating electron transfer. This study underscores the necessity for comprehensive computational strategies to analyze enzyme-polymer interactions beyond simplistic models, uncovering critical binding determinants and informing future mutagenesis experiments, in order to enhance laccase performance and rationalize variations in enzymatic activity.

AbstractThe thermodynamics and kinetics of heterogeneous electron transfer (ET) for Co‐substituted horse myoglobin (Co−Mb) and its derivatives with ammonia and imidazole as heme axial ligands were studied with cyclic voltammetry on a pyrolytic graphite electrode along with their ability to mediate the electro‐catalytic production of H2. All the proteins experience a non‐diffusive electrochemical regime as electrode‐bound species. The adsorbed Co−Mb construct carries out the electro‐catalytic reduction of water protons to H2 with a good efficiency under anaerobic conditions thus yielding a simple and tunable system for H2 production. Replacement of H2O as Co axial ligand by ammonia and imidazole significantly lowers the catalytic currents for H3O+/H2O reduction to H2. The E°’ values of the Co(III)/Co(II) redox couple for all species are mainly determined by the enthalpic contribution. Differences were found in the kinetics of ET for the different protein adducts due to changes in the activation enthalpies. However, all species share the same distance of about 14 Å from the electrode surface to the Co(III)/Co(II) center determined using the Marcus model, consistent with a non‐denaturing adsorption of the protein.

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered in the past decade. Their importance in the degradation of recalcitrant substrates in sustainable processes is now well established; however, the catalytic peroxygenase mechanism has yet to be fully understood. This is also because the study of reaction kinetics has to deal with multiple variables, including the nature of the substrates, the presence of unwanted side reactions, and the low protein stability in the presence of H2O2 as the cosubstrate. In this work, three bacterial LPMOs from the AA10 family were investigated: Pseudomonas putida AA10 (PpAA10), Streptomyces coelicolor AA10B (ScAA10B), and AA10C (ScAA10C). Their activity against specific substrates was initially evaluated by ATR-FTIR for a qualitative characterization, and then, to determine electrochemically their kinetic constants, an amperometric assay based on the detection of the H2O2 consumption was used. This allowed the determination of turnover numbers (TNs) and total turnover numbers (TTNs) on different substrates. The best performance was obtained with ScAA10C and ScAAA10B on nanocrystalline cellulose with TNs of 3.81 and 2.88 s(-1), respectively, and TTNs of 1208 and 735, respectively. PpAA10 is active on beta-chitin with a TN of 1.02 s(-1) and a TTN of 61, providing valuable insight into their substrate specificity and stability. Although the initial rates were found to be lower than those for fungal LPMOs, the enzyme stability over time increased on more crystalline substrates and in the presence of the carbohydrate-binding module (CBM), yielding activity values comparable to those for fungal LPMOs. Moreover, the presence of the CBM resulted in a more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and increased resistance to oxidative inactivation.

The cleavage of the axial S(Met) - Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycer-ophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV - VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe -M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in com-parison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467-487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe - S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt species.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

In the present study, human neuroglobin (hNgb) was found to undergo H2O2‐induced breakdown of the heme center at a much slower rate than other globins, namely in the timescale of hours against minutes. We investigated how the rate of the process is affected by the Cys46/Cys55 disulfide bond and the network of non‐covalent interactions in the distal heme side involving Tyr44, Lys67, the His64 heme iron axial ligand and the heme propionate‐7. The rate is increased by the Tyr44 to Ala and Phe mutations; however the rate is lowered by Lys67 to Ala swapping. The absence of the disulfide bridge slows down the reaction further. Therefore, the disulfide bond‐controlled accessibility of the heme site and the residues at position 44 and 67 affect the activation barrier of the reaction. Wild‐type and mutated species form β‐amyloid aggregates in the presence of H2O2 producing globular structures. Furthermore, the C46A/C55A, Y44A, Y44F and Y44F/C46A/C55A variants yield potentially harmful fibrils. Finally, the nucleation and growth kinetics for the aggregation of the amyloid structures can be successfully described by the Finke–Watzky model.

Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics.

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.

The autosomal dominant striated muscle disease myoglobinopathy is due to the single point mutation His98Tyr in human myoglobin (MB), the heme protein responsible for binding, storage, and controlled release of O2 in striated muscle. In order to understand the molecular basis of this disease, a comprehensive biochemical and biophysical study on wt MB and the variant H98Y has been performed. Although only small differences exist between the active site architectures of the two proteins, the mutant (a) exhibits an increased reactivity toward hydrogen peroxide, (b) exhibits a higher tendency to form high‐molecular‐weight aggregates, and (c) is more prone to heme bleaching, possibly as a consequence of the observed H2O2‐induced formation of the Tyr98 radical close to the metal center. These effects add to the impaired oxygen binding capacity and faster heme dissociation of the H98Y variant compared with wt MB. As the above effects result from bond formation/cleavage events occurring at the distal and proximal heme sites, it appears that the molecular determinants of the disease are localized there. These findings set the basis for clarifying the onset of the cascade of chemical events that are responsible for the pathological symptoms of myoglobinopathy.

The thermodynamic and kinetic properties for heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccinimide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°′ values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can, respectively, be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°′ values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox = 5.1, pKa1red = 7.5, pKa2ox = 8.4, pKa2red = 10.9, pKa2ox = 8.9, pKa2red = 11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°′ value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and medium conditions.

In this study, eight lactic acid bacteria (LAB) strains, previously isolated from traditional and gluten-free sourdoughs, and selected for their potential in improving the sensory and rheological quality of bakery products, were screened against some common spoilage agents. The anti-mould activity was tested using strains of the species Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger. Regarding the antibacterial activity, it was assessed against four strains of the species Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes. Furthermore, LAB strains were evaluated for their ability to produce exopolysaccharides, which are gaining considerable attention for their functional properties and applicability in different food industrial applications. A strain-specific behaviour against the moulds was observed. In particular, F. graminearum ITEM 5356 was completely inhibited by all the LAB strains. Regarding the antibacterial activity, the strains Leuconostoc citreum UMCC 3011, Lactiplantibacillus plantarum UMCC 2996, and Pediococcus pentosaceus UMCC 3010 showed wide activity against the tested pathogens. Moreover, all the LAB strains were able to produce exopolysaccharides, which were preliminarily characterized. The assessed features of the LAB strains allow us to consider them as promising candidates for single or multiple starter cultures for food fermentation processes.

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

The electrode-immobilized Met80Ala variant of yeast iso-1 cytochrome c in mixed water/dimethylsulfoxide (DMSO) solutions up to 60 % v/v DMSO shows thermodynamic and kinetic parameters of electron exchange and electrocatalytic properties towards O-2 reduction fully comparable to those in water. This is the result of moderate protein conformational changes thanks to immobilization that, to a certain extent, preserves protein structure, possibly due to the constraints on protein mobility/flexibility induced by the electrostatic interactions with the electrode-coating SAM. Upon increasing the DMSO content of the mixed solution beyond 60 %, a much larger perturbation occurs that leads to the progressive loss of the electrocatalytic ability. Therefore, under these conditions, the organic solvent remarkably affects the structure and properties of the protein probably involving major conformational changes or even the replacement of the 6(th) axial hydroxide ligand of the heme iron with a strong protein ligand, possibly a lysine residue.

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.

Splitting the reduction potential of electron transport (ET) proteins and redox metalloenzymes into the enthalpic and entropic contributions is an insightful practice to gaining insight into the molecular determinants of the thermodynamic propensity of the metal center to accept or release electrons. The strict control of such propensity is essential for the functioning of the electron transport chains in bioenergetics and the organized network of the countless reactions of the redox metabolism in all organisms. Here, the first comprehensive overview is offered on the thermodynamic data obtained in the last three decades for the main classes of ET species, namely c-type cytochromes and proteins containing T1 copper and ironsulfur centers, along with some heme metalloenzymes. These families show many common features in the balance of the enthalpic and entropic terms, which will be brought to light. The enthalpic terms related to ligation features in the first coordination sphere of the metal and weak binding and electrostatics in the surrounding matrix do count a lot in this balance. Reduction entropy is much less important that it would appear from the raw thermodynamic data, particularly for electron transport (ET) metalloproteins. This is due to reduction-induced solvent-related molecular events which dominate the measured entropy changes but affect much less the reduction free energy due to the compensatory effects of the associated enthalpic terms (a phenomenology known as enthalpy-entropy compensation, EEC). Thus the entropy changes seldom exert a real influence on the E degrees' of metalloredox proteins; this is restricted to metal sites subjected to reduction-induced protein-based changes in the accessible configurational microstates. It follows that in most cases, especially for ET species, the E degrees changes due to point mutations, ligand binding and charge changes have an ultimate enthalpic origin. Hence, they should be accounted for with coordination chemistry and electrostatics notions. Only if they don't, protein-based entropic effects could play a role. In this review, we go through the data gathered for the main classes of ET species and heme enzymes that brought us to this conclusion. (C) 2021 Elsevier B.V. All rights reserved.