Site Of Enzyme Research Articles

Electrospun Membranes Loaded with Melanin Derived from Pecan Nutshell (Carya illinoinensis) Residues for Skin-Care Applications

This study investigates the incorporation of melanin extracted from pecan nutshell residues into a polyacrylonitrile (PAN) matrix during the electrospinning of microfiber membranes. Melanin concentrations of 0.5, 2.0, and 5.0% w/w were incorporated to enhance the physicochemical and biological properties of the fibers. The melanin-loaded PAN fibers exhibited significant antioxidant activity against DPPH and ABTS radicals, with scavenging rates ranging from 46.58% to 62.77% and 41.02% to 82.36%, respectively, while unmodified PAN fibers showed no activity. Furthermore, the melanin-loaded membranes demonstrated antimicrobial effects. The membranes also exhibited an important enzyme inhibition activity against collagenase (37%), hyaluronidase (22%), tyrosinase (36%), and elastase (33%). Molecular docking studies reveal different potential amino acids of the active sites of aging enzymes that interact strongly with melanin pigment, particularly collagenase, followed by hyaluronidase, tyrosinase, and elastase. These results suggest that the novel melanin-loaded PAN membranes possess promising bioactive properties with potential applications in different skin-care applications.

Iron-sulfur clusters: the road to room temperature.

Iron-sulfur proteins perform a wide variety of reactions central to the metabolisms of all living organisms. Foundational to their reaction chemistry are the rich electronic structures of their constituent Fe-S clusters, which differ in important ways from the active sites of mononuclear Fe enzymes. In this perspective, we summarize the essential electronic structure features that make Fe-S clusters unique, and point to the need for studies aimed at understanding the electronic basis for their reactivity under physiological conditions. Specifically, at ambient temperature, both the ground state and a large number of excited states are thermally populated, and thus a complete understanding of Fe-S cluster reactivity must take into account the properties, energies, and reactivity patterns of these excited states. We highlight prior research toward characterizing the low-energy excited states of Fe-S clusters that has established what is now a consensus model of these excited state manifolds and the bonding interactions that give rise to them. In particular, we discuss the low-energy alternate spin states and valence electron configurations that occur in Fe-S clusters of varying nuclearities, and finally suggest that there may be unrecognized functional roles for these states.

Identification of HIV-1 Reverse Transcriptase-Associated Ribonuclease H Inhibitors Based on 2-Hydroxy-1,4-naphthoquinone Mannich Bases

There is a strong demand for new and efficient antiviral compounds. A series of 2-hydroxy-1,4-naphthoquinone Mannich bases were screened for their HIV-1-RNase H inhibitory activity. An HIV-1-RNase H assay was used to study the RNase H inhibition by the test compounds. Docking of active derivatives into the active site of the enzyme was carried out. Compounds 1e and 2k showed distinctly higher HIV-1-RNase H inhibitory activity (IC50 = 2.8–3.1 µM) than the known inhibitors RDS1759 and compound 13. The binding mode and possible interactions of 1e and 2k with the HIV-1-RNase H active site were determined using molecular docking, which led to the identification of salient and concealed pharmacophoric features of these molecules. The docking analysis revealed that there are significant differences in the binding mode of these compounds within the active site of the target enzyme. A selection of HIV-1-RNase H-inhibitory Mannich bases was tested for antiviral activity against HIV-1, and compound 2k showed the highest activity at low toxicity to host cells. The lawsone Mannich bases 1e and 2k also underwent a preliminary screening for activity against SARS-CoV-2, and compound 1e was found to inhibit SARS-CoV-2 replication (IC50 = 11.2 µM).

Evaluating the potential of Kalanchoe pinnata, Piper amalago amalago, and other botanicals as economical insecticidal synergists against Anopheles gambiae

BackgroundSynergists reduce insecticide metabolism in mosquitoes by competing with insecticides for the active sites of metabolic enzymes, such as cytochrome P450s (CYPs). This increases the availability of the insecticide at its specific target site. The combination of both insecticides and synergists increases the toxicity of the mixture. Given the demonstrated resistance to the classical insecticides in numerous Anopheles spp., the use of synergists is becoming increasingly pertinent. Tropical plants synthesize diverse phytochemicals, presenting a repository of potential synergists.MethodsExtracts prepared from medicinal plants found in Jamaica were screened against recombinant Anopheles gambiae CYP6M2 and CYP6P3, and Anopheles funestus CYP6P9a, CYPs associated with anopheline resistance to pyrethroids and several other insecticide classes. The toxicity of these extracts alone or as synergists, was evaluated using bottle bioassays with the insecticide permethrin. RNA sequencing and in silico modelling were used to determine the mode of action of the extracts.ResultsAqueous extracts of Piper amalago var. amalago inhibited CYP6P9a, CYP6M2, and CYP6P3 with IC50s of 2.61 ± 0.17, 4.3 ± 0.42, and 5.84 ± 0.42 μg/ml, respectively, while extracts of Kalanchoe pinnata, inhibited CYP6M2 with an IC50 of 3.52 ± 0.68 μg/ml. Ethanol extracts of P. amalago var. amalago and K. pinnata displayed dose-dependent insecticidal activity against An. gambiae, with LD50s of 368.42 and 282.37 ng/mosquito, respectively. Additionally, An. gambiae pretreated with K. pinnata (dose: 1.43 μg/mosquito) demonstrated increased susceptibility (83.19 ± 6.14%) to permethrin in a bottle bioassay at 30 min compared to the permethrin only treatment (0% mortality). RNA sequencing demonstrated gene modulation for CYP genes in anopheline mosquitoes exposed to 715 ng of ethanolic plant extract at 24 h. In silico modelling showed good binding affinity between CYPs and the plants’ secondary metabolites.ConclusionThis study demonstrates that extracts from P. amalago var. amalago and K. pinnata, with inhibitory properties, IC50 < 6.95 μg/ml, against recombinant anopheline CYPs may be developed as natural synergists against anopheline mosquitoes. Novel synergists can help to overcome metabolic resistance to insecticides, which is increasingly reported in malaria vectors.Graphical

An Active-Site Bro̷nsted Acid-Base Catalyst Destabilizes Mandelate Racemase and Related Subgroup Enzymes: Implications for Catalysis.

Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction, i.e., the metal-assisted, Bro̷nsted base-catalyzed abstraction of the α-proton from a carboxylate substrate to form an enol(ate) intermediate. Although the catalytic machinery responsible for the initial deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. Using differential scanning calorimetry, we examined the contribution of the Bro̷nsted acid-base catalysts to the thermostability (Tm) of four members of the mandelate racemase (MR)-subgroup of the ENS: MR, d-tartrate dehydratase, l-talarate/galactarate dehydratase, and l-fuconate dehydratase. Each enzyme contains an active-site Lys (part of a KxK motif) and His, which act as Bro̷nsted acid-base catalysts. The KxK → KxM substitutions increased the thermostability in all four enzymes with the effect being most prominent for MR (ΔTm = +8.6 °C). The KxK → MxK substitutions decreased the thermostability in all four enzymes, and the His → Asn substitution had a significant stabilizing effect only on MR. Thus, the active sites of MR-subgroup enzymes are destabilized by the Lys Bro̷nsted acid-base catalyst, suggesting that the destabilization energy may be used to drive a conformational change of the enzyme to yield a catalytically competent protonation state upon substrate binding.

Theoretical Study of Sphingomyelinases from Entamoeba histolytica and Trichomonas vaginalis Sheds Light on the Evolution of Enzymes Needed for Survival and Colonization.

The path to survival for pathogenic organisms is not straightforward. Pathogens require a set of enzymes for tissue damage generation and to obtain nourishment, as well as a toolbox full of alternatives to bypass host defense mechanisms. Our group has shown that the parasitic protist Entamoeba histolytica encodes for 14 sphingomyelinases (SMases); one of them (acid sphingomyelinase 6, aSMase6) is involved in repairing membrane damage and exhibits hemolytic activity. The enzymatic characterization of aSMase6 has been shown to be activated by magnesium ions but not by zinc, as shown for the human aSMase, and is strongly inhibited by cobalt. However, no structural data are available for the aSMase6 enzyme. In this work, bioinformatic analyses showed that the protist aSMases are diverse enzymes, are evolutionarily related to hemolysins derived from bacteria, and showed a similar overall structure as parasitic, free-living protists and mammalian enzymes. AlphaFold3 models predicted the occupancy of cobalt ions in the active site of the aSMase6 enzyme. Cavity blind docking showed that the substrate is pushed outward of the active site when cobalt is bound instead of magnesium ions. Additionally, the structural models of the aSMase6 of E. histolytica showed a loop that is absent from the rest of the aSMases, suggesting that it may be involved in hemolytic activity, as demonstrated experimentally using the recombinant proteins of aSMase4 and aSMase6. Trichomonas vaginalis enzymes show a putative transmembrane domain and seem functionally different from E. histolytica. This work provides insight into the future biochemical analyses that can show mechanistic features of parasitic protists sphingomyelinases, ultimately rendering these enzymes potential therapeutic targets.

Accurate sequence-to-affinity models for SH2 domains from multi-round peptide binding assays coupled with free-energy regression.

Short linear peptide motifs play important roles in cell signaling. They can act as modification sites for enzymes and as recognition sites for peptide binding domains. SH2 domains bind specifically to tyrosine-phosphorylated proteins, with the affinity of the interaction depending strongly on the flanking sequence. Quantifying this sequence specificity is critical for deciphering phosphotyrosine-dependent signaling networks. In recent years, protein display technologies and deep sequencing have allowed researchers to profile SH2 domain binding across thousands of candidate ligands. Here, we present a concerted experimental and computational strategy that improves the predictive power of SH2 specificity profiling. Through multi-round affinity selection and deep sequencing with large randomized phosphopeptide libraries, we produce suitable data to train an additive binding free energy model that covers the full theoretical ligand sequence space. Our models can be used to predict signaling network connectivity and the impact of missense variants in phosphoproteins on SH2 binding.

Purification, characterization, optimization, and docking simulation of alkaline protease produced by Brevibacillus agri SAR25 using fish wastes as a substrate.

Recycling of protein-rich environmental wastes and obtaining more valuable products from these recycled products is a topic of interest for researchers. This study aims to produce, purify, and characterize the physicochemical and structural properties of the protease enzyme produced from Brevibacillus agri SAR25 using salmon fish waste as substrate and also to evaluate the effect of protease on the chicken feather, enzyme-ligand interactions, and active site surface area. The production of protease was optimum on 50g/L fish waste, pH8, 40°C, 96h, and 150rpm. The alkaline protease was isolated using three-phase partitioning (TPP), a straightforward and efficient one-step method for purifying enzymes in industrial applications. TPP achieved a purification efficiency of 115% with a 3.1-fold increase in concentration. As a result, the molecular weight of the purified protease was determined to be 50kDa under optimal conditions of pH9 and 45°C. The 3D structure of the alkaline protease enzyme with the determined ligands was predicted by homology modeling using UCSF Chimera 1.17.3 software. Tween-20 ligand showed the best binding affinity by hydrogen bonding with amino acids 106A, Asn 133A, Gly 198, Asn 226, Glu 232 and hydrophobic interaction with His 135, Leu 283 in the active site of the alkaline protease enzyme, thus leading to the discovery of new semisynthetic enzymes of salmon fish waste. The activity and stability of Brevibacillus agri SAR25 alkaline protease over a wide range of temperatures and pH, and its relatively high stability in the presence of various metal ions, organic solvents, and surfactants indicate that it can be used as a biocatalyst for different industrial applications. This could lead to the creation of new enzymes made from recycled biological materials.

A potential therapeutic strategy by fungal laccase targeting novel binding sites on human cytomegalovirus DNA polymerase.

Human cytomegalovirus (HCMV) is a common herpesvirus that can severely affect transplant recipients, those with AIDS, and newborns. Existing synthetic medications face limitations, including toxicity, processing issues, and viral resistance. As part of this study, the efficacy of the extracellular enzyme laccase isolated from a widely available mushroom (Pleurotus pulmonarius) was compared to that of ganciclovir, a common antiviral, used against HCMV. The study found that laccase can synergistically inhibit HCMV replication by targeting new inhibitory sites on the UL54 protein. Viral replication requires significant energy, increasing cellular respiration. The antiviral effect of laccase was linked to reduced expression of genes regulating cellular respiration, which coincided with decreased viral DNA copies. Additionally, in silico analysis has identified a novel binding site for the laccase enzyme in the C-terminal region of HCMV DNA polymerase specifically between amino acids 1004 and 1242, which effectively obstructs the binding of the essential viral replication regulatory accessory protein UL44, thereby hindering successful replication. Molecular dynamics simulations were performed under standardized conditions mimicking a cellular environment, revealing a stable protein-protein docking complex. This study may aid in developing novel antiviral strategies by utilizing laccase's target specificity to regulate host cellular pathways against Herpesviridae.

Implications of non-native metal substitution in carbonic anhydrase - engineered enzymes and models.

The enzyme carbonic anhydrase has been intensely studied over decades as a means to understand the role of zinc in hydrating CO2. The naturally occurring enzyme has also been immobilized on distinct heterogeneous platforms, which results in a different hybrid class of catalysts that are useful for the adsorption and hydration of CO2. However, the reusability and robustness of such natural and immobilized systems are substantially affected when tested under industrial conditions, such as high temperature and high flow rate. This led to the generation of model systems in the form of metal-coordination complexes, metal-organic frameworks, metallo-peptide self-assembled supramolecules and nanomaterials that mimic the primary, and, to some extent, secondary coordination sphere of the active site of the natural carbonic anhydrase enzymes. Furthermore, the effects of zinc-substitution by other relevant transition metals in both the naturally occurring enzymes and model systems has been reported. It has been observed that some other transition metal ions in the active site of carbonic anhydrase and its models can also accomplish similar activity, established by various reaction probes and ideas. Herein, we present a comprehensive highlight about substituting zinc in the active site of the modified enzymes and its biomimetic model systems with non-native metal ions and review how they affect the structural orientation and reactivity towards CO2 hydration. In addition, the utility of artificially engineered carbonic anhydrases towards a number of non-natural reactions is also discussed.

Bifunctional chimeras of myeloperoxidase and glucose oxidase. Antimicrobial, topological and enzymatic properties.

Enhancing the local substrate concentration is a crucial strategy in nature for facilitating the proximity of two enzymes. The substrate of the first enzyme is transformed into a by-product that travels to the active site of the second enzyme without external diffusion, then transformed into a product and eventually expelled from the complex. In an effort to optimize the antimicrobial properties of myeloperoxidase from Rhodopirellula baltica (RbMPO), we created a library of fused chimeras between a glucose oxidase (GOx) and RbMPO so that H2O2 could be continuously perfused in the vicinity RbMPO, enabling the production of HOCl or HOSCN, well-known antimicrobial agents. The enzymes were characterized biochemically, enzymatically, and physically using low-resolution techniques such as AFM, SAXS, and cryofracture. SAXS experiments revealed that the chimeras were properly folded and existed in different oligomeric states. The kinetic parameters of the chimeras were determined and used for classification, revealing that all chimeras exhibited varying levels of activity and were microbicidal. The mixture of different oligomeric states of LEGGEAEA displayed both activity and microbicidal properties. AFM was used to visualize the chimeras in different oligomeric states, with their overall shapes ranging from round, oblong, to hooked, depending on the linker used.

Assessment of the potential toxic of naringenin nanoparticles using ex vivo and in silico models.

Naringenin is a flavonoid known for its anti-inflammatory, antineoplastic, antiatherogenic, and antioxidant properties. However, it has poor technological characteristics and limited bioavailability, which hinder its use in food applications. Nanoencapsulation could address these limitations, but safety concerns regarding nanoengineered bioactives need to be resolved before they can be effectively utilized as food additives. The objective of this study was to evaluate the potential cytotoxic, genotoxic, and mutagenic effects of both free and encapsulated naringenin through in vivo experiments using Allium cepa L. roots, along with pharmacokinetic and molecular docking analyses. The results showed that naringenin nanoparticles did not produce significant changes in the cell division index of meristematic cells in A. cepa roots. Additionally, no significant alterations in the mitotic spindle or chromosomal breaks were observed. Molecular docking studies indicated that naringenin effectively binds to the active site of the catalase enzyme (CAT) in a competitive manner, while it attaches to a site away from the active site of superoxide dismutase (SOD2), demonstrating a non-competitive interaction. ADMET property assessments suggested that naringenin exhibits relatively low toxicity and has favorable molecular characteristics for oral administration. In summary, this study supports the potential of naringenin, particularly in its nanoencapsulated form, as a safe and effective ingredient for functional foods, provided that safety concerns regarding nanoencapsulation are adequately addressed.

Identification of new inhibitors of Plasmodium falciparum hypoxanthine–guanine–xanthine Phosphoribosyltransferase (HG(X)PRT): An outlook towards the treatment of malaria

Plasmodium, a protozoan parasite responsible for causing malaria relies on the purine salvage pathway to synthesize purine as they are incapable of synthesizing them de novo. This pathway is crucial for the survival of the parasite and hence enzymes of this pathway can serve as antimalarial drug targets. One of the enzymes of this pathway is hypoxanthine guanine (xanthine) phosphoribosyltransferase [HG(X)PRT] that serves as novel target, potentially less prone to existing resistance mechanisms seen with the use of traditional antimalarial drugs. HGXPRT inhibition disrupts the parasite's ability to synthesize nucleotides, essential for its growth and replication. In this regard, the current study was designed to identify the inhibitors of HGXPRT. For this purpose, the enzyme was produced through recombinant technology and purified with 10 mg/ L yield. Followed this, UV-based enzyme inhibition assay was optimized and >200 fully characterized compounds were evaluated for their HGXPRT inhibitory activity. Out of them fourteen compounds 1–14 showed significant to weak inhibition of HGXPRT enzyme with IC50 values in the range of 15.7 to 229.6 μM, as compared to the standard inhibitor i.e. 9-deazaguanine (IC50 = 12 ± 1.0 μM). In- silico and biophysical studies were further performed on active compounds to get structural insights into enzyme-inhibitor complex at the atomic level. Docking studies predicted that these inhibitors accommodate the purine binding site of enzyme and interacted with critical residues such as Asp148, Phe197, and Val198. Biophysical studies showed that these identified inhibitors interacted with HGXPRT enzyme in a non-ambiguous manner. Furthermore, these inhibitors were found to be non-cytotoxic against human fibroblast cell lines (BJ). Hence, this study identified 14 hits that could lead to further research towards anti-malarial drug design and development.

Rational pore engineering reveals the relative contribution of enzymatic sites and self-assembly towards rapid ferroxidase activity and mineralization: impact of electrostatic guiding and cage-confinement in bacterioferritin.

The self-assembled ferritin protein nanocage plays a pivotal role during oxidative stress, iron metabolism, and host-pathogen interaction by executing rapid iron uptake, oxidation and its safe-storage. Self-assembly creates a nanocompartment and various pores/channels for the uptake of charged substrates (Fe2+) and develops a concentration gradient across the protein shell. This phenomenon fuels rapid ferroxidase activity by an upsurge in the substrate concentration at the catalytic sites. However, it is difficult to segregate the relative contributions of the catalytic sites and self-assembly towards rapid ferroxidase/mineralization activity owing to the inherent self-assembly propensity of ferritins. In the current work, 3-fold pore electrostatics of bacterioferritin from Mycobacterium tuberculosis were rationally altered by site-directed mutagenesis to generate self-assembled (E121A and E121Q) and assembly-defective (E121K and E121F) variants. In comparison to the autoxidation of Fe2+ in buffer, the assembly-defective variants exhibited significantly faster ferroxidase/mineralization activity and O2 consumption kinetics due to their functional catalytic sites, but failed to level-up with the self-assembled variants even at 100-fold higher Fe2+ concentration. Only the self-assembled variants exhibited cooperativity in iron oxidation, maintained biomineral solubility, and protected DNA against the Fenton reaction. This report highlights the concerted effect of self-assembly and ferroxidase sites that propels the rapid Fe2+ uptake, its oxidation and biomineralization in bacterioferritin. The findings also establish the importance of electrostatic guiding and nanoconfinement offered by ferritin self-assembly towards its enzymatic activity and antioxidative properties. Moreover, this work identifies the key electrostatic interactions ("hot-spots") at the subunit contact points that control the cage/pore formation, impart cage stability and influence ferritin's natural functions. Manipulation of hot-spot residues can be further extended towards the encapsulation of cargo, for various bio-medical applications, by strategically inducing its disassembly and subsequent reassembly through adjustments in ionic strength. This would bypass the need for extreme/harsh reaction conditions and minimize the loss of cargo/protein.

Two New α-Glucosidase Inhibitors from Haplophyllum tuberculatum: Inhibition Kinetics and Mechanistic Insights Through in Vitro and in Silico Approaches.

Diabetes is a multifactorial global health disorder marked by unusually high plasma glucose levels, which can lead to serious consequences including diabetic neuropathy, kidney damage, retinopathy, and cardiovascular disease. One effective therapy approach for reducing hyperglycemia associated with type 2 diabetes is to target α-glucosidase, enzymes that catalyze starch breakdown in the intestine. In the current study, two new (1, 2) and nine known (3-11) compounds were isolated from the rutaceous plant Haplophyllum tuberculatum and characterized by extensive nuclear magnetic resonance spectroscopic techniques and high-resolution electrospray ionization mass spectrometry. After structural elucidation, nine compounds were evaluated for their ability to inhibit α-glucosidase, a target for the treatment of type-2 diabetes. Among them, three compounds (7, 5, and 2) exhibited notable inhibition with half-maximal inhibitory concentration (IC50) values of 3.42 ± 0.12, 5.79 ± 0.28, and 6.75 ± 1.18µM, respectively, while the remaining six compounds (1, 3, 4, 6, 8, and 9) had a moderate activity with IC50 values ranging from 12.14 ± 0.35 to 24.60 ± 0.57µM, compared to the standard drug acarbose (IC50 = 875.75 ± 1.24µM). A kinetic study of compounds 5 and 7 exhibited the competitive type of inhibition with Ki values of 4.82 ± 0.0036 and 3.92 ± 0.0062µM, respectively. Furthermore, a structure-based prediction of the compounds' binding mode suggested that these inhibitors fitted exceptionally well within the active site of the target enzyme, α-glucosidase, forming multiple hydrogen and hydrophobic interactions with its active site residues. In conclusion, compounds with potent α-glucosidase inhibitory activity are abundant in nature and can be explored and further developed for treating diabetes mellitus.

'Two in One' Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion-Ligation and Screening of Positive Recombinants by Unaided Eyes.

To clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. Some cloning vectors have been developed for the cloning of PCR products, including blunt-end and T-A cloning. However, different plasmids are required for the cloning of PCR products with blunt ends and 3' A overhang ends. Here, a novel cloning vector, pYFRed, which is based on the pUC19 backbone, has emerged and can be applied in both blunt-end and T-A cloning. PCR products can be cloned into the pYFRed by a one-step digestion-ligation reaction in a tube. The endonuclease recognition sequences of SmaI, Eco53kI, EcoRV, PmeI, and SwaI for blunt-end cloning and XcmI for T-A cloning were designed and added between the lac promoter and the starting codon ATG of the mScarlet-I gene of pYFRed. The ligation efficiency was significantly higher because the restriction enzyme sites utilized were removed from the vector after being successfully constructed. The mScarlet-I gene was introduced into the pYFRed for the screening of the positive recombinants by the unaided eye without the need for additional reagents/equipment. pYFRed is easy to construct in an ordinary laboratory, which facilitates researchers to develop their cloning vector without purchasing commercial cloning vectors.

In Silico Study Highlighting the Interactions Between the Active Sites of Steroidogenic Enzymes and Endocrine Disruptors Found in Daily Use Products

In Silico Study Highlighting the Interactions Between the Active Sites of Steroidogenic Enzymes and Endocrine Disruptors Found in Daily Use Products

Investigating the Antibacterial Effects of Cuminum cyminum and Foeniculum vulgare Essential Oils on Escherichia coli and Listeria monocytogenes

Background: Due to the increasing demand for new antibiotics, extensive research is being conducted on various compounds, particularly plant-based compounds. Among them, plant essential oils (EOs) have been investigated for their antibacterial properties in numerous studies. Accordingly, the inhibitory and bactericidal effects of Cuminum cyminum and Foeniculum vulgare EOs on two bacterial species, Escherichia coli and Listeria monocytogenes, were examined in this study. Methods: The compounds present in these extracts were evaluated using gas chromatography-mass spectrometry analysis. Subsequently, molecular docking methods were employed to depict the interactions of these compounds with the active site of the beta-lactamase enzyme of these bacteria in three-dimensional illustrations. Results: Laboratory methods such as disk diffusion agar, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) revealed that although the bactericidal properties of these extracts were weak, they exhibited strong growth inhibitory properties, particularly the effect of F. vulgare EO on L. monocytogenes (MIC=1/104). Additionally, with the presence of p-cumin aldehyde at 33.7% in the cumin extract and trans-anethole at 46.5% in the fennel extract, molecular docking analyses showed that the binding ability and antibacterial properties of the cumin extract against E. coli by two hydrogen bonds and fennel extract against L. monocytogenes by two hydrogen bonds were more pronounced considering the level of binding energies. Conclusion: Although the extracts represented weak bactericidal properties, their strong inhibitory effects on bacterial growth, especially with F. vulgare EO on L. monocytogenes, are notable. Moreover, the active compounds, such as p-cumin aldehyde and trans-anethole, demonstrated significant potential as alternative antibacterial agents.

Molecular modeling, synthesis and biological evaluation of caffeic acid based Dihydrofolate reductase inhibitors

Dihydrofolate reductase (DHFR) is an enzyme that plays a crucial role in folate metabolism, which is essential for cell growth and division. DHFR has been identified as a molecular target for numerous diseases due to its significance in various biological processes. DHFR inhibitors can disrupt folate metabolism by inhibiting DHFR, leading to the inhibition of cell growth. So, a series of caffeic acid derivatives were designed, synthesized, characterized and evaluated for their in vitro ability to inhibit DHFR, as well as their antimicrobial and anticancer properties. Among all synthesized compounds, compound CE11 exhibited the highest DHFR inhibitory activity, with an IC50 value of 0.048 µM, which is approximately four times more potent than methotrexate. Compound CE11 exhibited similar docking performance to methotrexate, binding to the same site and engaging key residues such as Glh30, Phe31, Phe34, and Ser59. It also fit snugly in the hydrophobic pocket of modeled protein. Moreover, substantial hydrophobic interactions were noted between the ligand and the hydrophobic amino acid residues of DHFR. This effectively secured the derivative within the restricted substrate cavity. Furthermore, compound CE11 demonstrated a significant anticancer activity against MCF-7 breast cancer cell line, with an IC50 value of 5.37 ± 0.16 µM. Compounds CE3 and CE15 displayed better antibacterial activity compared to trimethoprim and comparable to ampicillin against the gram-positive bacteria S. aureus. Moreover, compounds CE3 and CE15 have shown better antibacterial activity than standard trimethoprim, ampicillin and tetracycline against the gram-negative bacteria, particularly P. aeruginosa and E. coli. Molecular docking analysis of CE3 revealed that it firmly entrapped into the active site of enzyme through hydrophobic interaction with hydrophobic residues. Additionally, it forms hydrogen bonds with important amino acid residues Ala7, Asn18, and Thr121 with excellent docking score and binding energy (-9.9, -71.77 kcal/mol). These interactions might be contributed to the significant DHFR inhibition and antimicrobial activity. The generated model holds potential value in facilitating the development of a novel category of DHFR inhibitors as anticancer and antimicrobial agents.

Ultra-sensitive fiber optic LSPR biosensor with enhanced enzyme functionalization for real-time putrescine detection.

Putrescine is a kind of physical diamine that is closely related to food deterioration and food quality safety. This study employs a novel fiber optic biosensor based on S-tapered and waist extension techniques, as well as localized surface plasmon resonance (LSPR), to detect putrescine accurately. The gold nanoparticles (AuNPs) are fixed on the fiber to excite LSPR. Furthermore, the multi-walled carbon nanotubes (MWCNTs) and niobium carbide (Nb2CTx) are used to increase the binding sites of enzymes to improve the sensing performance of probes. In this work, the fiber probe surface is functionalized by diamine oxidase (DAO) to improve selectivity. The sensitivity and limit of detection of the fiber optic biosensor are 2.04 nm/lg(µM) and 0.267 µM over a linear range of 0-100 µM, respectively. In addition, the tests of repeatability, reusability, selectivity, pH value, and real samples of the sensor probe are assessed to demonstrate the potential in measuring putrescine.