On the antibacterial photodynamic inactivation mechanism of Emodin and Dermocybin natural photosensitizers: A theoretical investigation.

A DFT and TDDFT study has been carried out on monomeric anthraquinones Emodin and Dermocybin (Em, Derm) recently proposed as natural antibacterial photosensitizers able to act also against gram-negative microbes. The computational study has been performed considering the relative amount of neutral and ionic forms of each compound in water, with the variation of pH. The occurrence of both Type I and Type II photoreactions has been explored computing the absorption properties of each species, the spin-orbit coupling constants (SOC), the vertical ionization potentials and the vertical electron affinities. The most plausible deactivation channels leading to the population of excited triplet states have been proposed. Our data indicate Emodin as more active than Dermocybin in antimicrobial photodynamic therapy throughout the Type II mechanism. Our data support a dual TypeI/II activity of the monomeric anthraquinones Emodin and Dermccybin in water, in all the considered protonation states.

Viral RNA Replication Suppression of SARS-CoV-2: Atomistic Insights into Inhibition Mechanisms of RdRp Machinery by ddhCTP.

The nonstructural protein 12, known as RNA-dependent RNA polymerase (RdRp), is essential for both replication and repair of the viral genome. The RdRp of SARS-CoV-2 has been used as a promising candidate for drug development since the inception of the COVID-19 spread. In this work, we performed an in silico investigation on the insertion of the naturally modified pyrimidine nucleobase ddhCTP into the SARS-CoV-2 RdRp active site, in a comparative analysis with the natural one (CTP). The modification in ddhCTP involves the removal of the 3'-hydroxyl group that prevents the addition of subsequent nucleotides into the nascent strand, acting as an RNA chain terminator inhibitor. Quantum mechanical investigations helped to shed light on the mechanistic source of RdRp activity on the selected nucleobases, and comprehensive all-atom simulations provided insights about the structural rearrangements occurring in the active-site region when inorganic pyrophosphate (PPi) is formed. Subsequently, the intricate pathways for the release of PPi, the catalytic product of RdRp, were investigated using Umbrella Sampling simulations. The results are in line with the available experimental data and contribute to a more comprehensive point of view on such an important viral enzyme.

On the Role of Temperature in the Depolymerization of PET by FAST-PETase: An Atomistic Point of View on Possible Active Site Pre-Organization and Substrate-Destabilization Effects.

Enzyme FAST-PETase, recently obtained by a machine learning approach, can depolymerize poly(ethylene terephthalate) (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET-based materials. In this study, a model of PET was adopted to describe the substrate, and all-atoms classical molecular dynamics (MD) simulations on apo- and substrate-bound FAST-PETase were carried out at 30 and 50 °C to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis shed light on the interactions occurring between the FAST-PETase and 4PET at 50 °C, the optimal working conditions of the enzyme. Pre-organization of the enzyme active and binding sites has been highlighted, while MD simulations of FAST-PETase:4PET pointed out the occurrence of solvent-inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations was observed during MD simulations of the substrate alone in solution performed at 30, 50 and 150 °C. The analysis led us to propose that, at 50 °C, the FAST-PETase is pre-organized to bind the PET and that the interactions occurring in the binding site can promote a more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.

Degradation of Penicillinic Antibiotics and ß-Lactamase Enzymatic Catalysis in a Biomimetic Zn-Based Metal-Organic Framework.

Invited for the cover of this issue are Jesús Ferrando-Soria, Donatella Armentano, Antonio Leyva-Pérez, Emilio Pardo and co-workers at University of Valencia, Technical University of Valencia and University of Calabria. The image depicts the crystal structure of a novel ZnII biological metal-organic framework that mimics ß-lactamase enzymes. Read the full text of the article at 10.1002/chem.202301325.

Degradation of Penicillinic Antibiotics and ß-Lactamase Enzymatic Catalysis in a Biomimetic Zn-Based Metal-Organic Framework.

ß-Lactam antibiotics are one of the most commonly prescribed drugs to treat bacterial infections. However, their use has been somehow limited given the emergence of bacteria with resistance mechanisms, such as ß-lactamases, which inactivate them by degrading their four-membered ß-lactam rings. So, a total knowledge of the mechanisms governing the catalytic activity of ß-lactamases is required. Here, we report a novel Zn-based metal-organic framework (MOF, 1), possessing functional channels capable to accommodate and interact with antibiotics, which catalyze the selective hydrolysis of the penicillinic antibiotics amoxicillin and ceftriaxone. In particular, MOF 1 degrades, very efficiently, the four-membered ß-lactam ring of amoxicillin, acting as a ß-lactamase mimic, and expands the very limited number of MOFs capable to mimic catalytic enzymatic processes. Combined single-crystal X-ray diffraction (SCXRD) studies and density functional (DFT) calculations offer unique snapshots on the host-guest interactions established between amoxicillin and the functional channels of 1. This allows to propose a degradation mechanism based on the activation of a water molecule, promoted by a Zn-bridging hydroxyl group, concertedly to the nucleophilic attack to the carbonyl moiety and the cleaving of C-N bond of the lactam ring.

Periodicity of the Affinity of Lanmodulin for Trivalent Lanthanides and Actinides: Structural and Electronic Insights from Quantum Chemical Calculations.

Lanmodulin (LanM) is the first identified macrochelator that has naturally evolved to sequester ions of rare earth elements (REEs) such as Y and all lanthanides, reversibly. This natural protein showed a 106 times better affinity for lanthanide cations than for Ca, which is a naturally abundant and biologically relevant element. Recent experiments have shown that its metal ion binding activity can be further extended to some actinides, like Np, Pu, and Am. For this reason, it was thought that LanM could be adopted for the separation of REE ions and actinides, thus increasing the interest in its potential use for industry-oriented applications. In this work, a systematic study of the affinity of LanM for lanthanides and actinides has been carried out, taking into account all trivalent ions belonging to the 4f (from La to Lu) and 5f (from Ac to Lr) series, starting from their chemistry in solution. On the basis of a recently published nuclear magnetic resonance structure, a model of the LanM-binding site was built and a detailed structural and electronic description of initial aquo- and LanM-metal ion complexes was provided. The obtained binding energies are in agreement with the available experimental data. A possible reason that could explain the origin of the affinity of LanM for these metal ions is also discussed.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations.

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Sulphur- and Selenium-for-Oxygen Replacement as a Strategy to Obtain Dual Type I/Type II Photosensitizers for Photodynamic Therapy.

The effect on the photophysical properties of sulfur- and selenium-for-oxygen replacement in the skeleton of the oxo-4-dimethylaminonaphthalimide molecule (DMNP) has been explored at the density functional (DFT) level of theory. Structural parameters, excitation energies, singlet-triplet energy gaps (ΔES-T), and spin-orbit coupling constants (SOC) have been computed. The determined SOCs indicate an enhanced probability of intersystem crossing (ISC) in both the thio- and seleno-derivatives (SDMNP and SeDMNP, respectively) and, consequently, an enhancement of the singlet oxygen quantum yields. Inspection of Type I reactions reveals that the electron transfer mechanisms leading to the generation of superoxide is feasible for all the compounds, suggesting a dual Type I/Type II activity.

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations.

Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.

Antioxidant and copper-chelating power of new molecules suggested as multiple target agents against Alzheimer's disease. A theoretical comparative study.

In this study, the scavenging activity against OOH radicals and the copper-chelating ability of two new synthesized molecules (named L1 and L2) that can act as multiple target agents against Alzheimer's disease have been investigated at the density functional theory level. The pKa and molar fractions at physiological pH have been predicted. The main antioxidant reaction mechanisms in lipid-like and water environments have been considered and the relative rate constants determined. The copper-chelating ability of the two compounds has also been explored at different coordination sites and computing the complexation kinetic constants. Results show the L1 compound is a more effective radical scavenging and copper-chelating agent than L2.

Computational Study Reveals the Role of Water Molecules in the Inhibition Mechanism of LAT1 by 1,2,3-Dithiazoles.

The L-type amino acid transporter LAT1, involved in many biological processes including the overexpression of some tumors, is considered a potential pharmacological target. The 1,2,3-Dithiazole scaffold was predicted to inhibit LAT1 by the formation of an intermolecular disulfide bond with the thiolate group of cysteine(s). As a result of the identification of these irreversible covalent inhibitors, we decided to deeply investigate the recognition stage and the covalent interaction, characterizing the chemical structures of the selected ligands. With the aim to provide new insights into the access of the ligands to the binding pocket and to reveal the residues involved in the inhibition, we performed docking, molecular dynamics simulations, and density functional theory-based investigation of three 1,2,3-dithiazoles against LAT1. Our computational analysis further highlighted the crucial role played by water molecules in the inhibition mechanism. The results here presented are consistent with experimental observations and provide insights that can be helpful for the rational design of new-to-come LAT1's inhibitors.

How Lanthanide Ions Affect the Addition-Elimination Step of Methanol Dehydrogenases.

The recently discovered methanol dehydrogenase, XoxF, is a widespread enzyme used by methylotrophic bacteria to oxidize methanol for carbon and energy, and requires lanthanide ions for its activity. This enzyme represents an essential component of methanol utilization by both methanol- and methane-utilizing bacteria. The present investigation looks on the electronic, energetic and geometrical behavior of the methanol dehydrogenase from Methylacidiphilum fumariolicum SolV, which is strictly dependent on early lanthanide metals with +3 oxidation states, by examining enzyme-substrate complexes of all the lanthanides. We focus on the catalytic reaction mechanism of two methanol dehydrogenases having as cofactor europium and ytterbium belonging to the mid- and later- series of lanthanides, in comparison with the methanol dehydrogenase containing the cerium, one early lanthanide. Our results provide evidence for the influence of the lanthanide contraction effect in all the elementary steps of the catalytic reaction mechanism. This indication may prove useful for developing new catalytic machineries of enzymes that adopt new-to-nature transformations.

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study.

Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme-substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.

Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology.

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

The Catalytic Mechanism of Human Transketolase.

We have computationally determined the catalytic mechanism of human transketolase (hTK) using a cluster model approach and density functional theory calculations. We were able to determine all the relevant structures, bringing solid evidences to the proposed experimental mechanism, and to add important detail to the structure of the transition states and the energy profile associated with catalysis. Furthermore, we have established the existence of a crucial intermediate of the catalytic cycle, in agreement with experiments. The calculated data brought new insights to hTK's catalytic mechanism, providing free-energy values for the chemical reaction, as well as adding atomistic detail to the experimental mechanism.

Why hydroxy-proline improves the catalytic power of the peptidoglycan N-deacetylase enzyme: insight from theory.

Nature exploits different strategies for enhancing the catalytic activity of enzymes, often resorting to producing beneficial mutations. The case of post-translational proline hydroxylation mutation in the active site of polysaccharide deacetylase (PDA) Bc1960 from Bacillus cereus is an interesting example of how small chemical modifications can cause significant improvements in enzymatic activity. In the present study the deacetylation mechanism promoted by both OH-proline (2Hyp) and standard proline (Pro) containing PDA is investigated using density functional theory. Although the mechanism presented for the two examined enzymes is in agreement with protease catalysis in metalloenzymes, the analysis along the potential energy surface (PES) reveals that the intermediate and product benefit energetically from the presence of the hydroxyl group on the proline. Our calculations provide evidence that for PDA-2Hyp, the hydrogen bond network established by the -OH group on the Cα of the proline with its closest neighbors stabilizes the transition states and, consequently, the reaction takes advantage of this. These results further contribute towards explaining the different catalytic activity experimentally observed for the polysaccharide deacetylase enzymes.

Antioxidant Properties of the Vam3 Derivative of Resveratrol.

A considerable number of studies has shown that many constituents of foods derived from plants are effective and safe antioxidants. This explains the growing interest in natural antioxidants in food applications. The goal of this investigation was to evaluate the antioxidant properties of the Vam3, a resveratrol derivative, firstly isolated from ethanol extracts of Vitis amurensis Rupr as a secondary product, and to carry out a comparison with resveratrol and other phenolic compounds which are currently in the limelight all over the world due to their beneficial effects on the human body. The potential of Vam3 as an antioxidant was determined through the evaluation of some key thermodynamic parameters which are commonly used for this purpose and describe the antioxidant activity quite well. Various mechanisms through which antioxidants usually can carry out their action were also explored both in water and in apolar environment. The results indicated that Vam3 is an excellent candidate as a natural antioxidant.

How Can Methanol Dehydrogenase from Methylacidiphilum fumariolicum Work with the Alien CeIII Ion in the Active Center? A†Theoretical Study.

Lanthanides are an example of nonbiogenic metal species and have been widely used in crystallographic and spectroscopic studies to probe Mg2+ /Ca2+ binding sites in metalloproteins by replacing the native cofactor. Recently, a methanol dehydrogenase (MDH) enzyme containing cerium ion in the active site has been isolated from Methylacidiphilum fumariolicum bacterium. With the aim to highlight as metal ion substitution can be reflected in catalytic mechanism, a comparative DFT study between Ca- and Ce-MDH has been undertaken. The obtained potential energy surfaces (PES), for two considered reaction mechanisms (named A and B), indicate mechanism A (addition-elimination and protonation processes) as the favored for both the enzymes and show as the barrier for the rate-determining step of Ce-MDH requires 19.4 kcal mol-1 .

Reaction Mechanism of Low-Spin Iron(III)- and Cobalt(III)-Containing Nitrile Hydratases: A Quantum Mechanics Investigation.

To elucidate the catalytic mechanism of cobalt(III)-benzonitrile and iron(III)--pivalonitrile hydratases, we have performed at density functional level a study using the cluster model approach. Computations were made in a protein framework. Following the suggestions given in a recent work on the analogous enzyme Fe(III)-NHase, we have explored the feasibility of a new working mechanism of examined enzymes. According to our results, after the formation of enzyme substrate complex, the reaction evolves toward product in only three steps. The first one is the nucleophilic attack, led by the -OH group of the αCys113-S-OH on the nitrile carbon atom, followed by the amide formation and by the enzyme restoring phase that our computations indicate as the most expensive step from the energetic point of view in both catalytic processes.