Search Results (1,566)

The enzyme ABH2, one of nine human DNA dioxygenases of the AlkB family, belongs to the superfamily of Fe(II)/α-ketoglutarate-dependent dioxygenases and plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases. ABH2 has broad substrate specificity, directly oxidizing DNA damages such as N¹-methyladenine, N³-methylcytosine, 1,N⁶-ethenoadenine, 3,N⁴-ethenocytosine, and a number of others. In our investigation, we sought to uncover the subtleties of the mechanisms governing substrate specificity in ABH2 by focusing on several critical amino acid residues situated in its active site. To gain insight into the function of this enzyme, we performed a functional mapping of its active site region, concentrating on pivotal residues, participating in forming a damaged binding pocket of the enzyme (Val99 and Ser125), as well as the residues directly involved in interactions with damaged bases, namely Arg110, Phe124, Arg172, and Glu175. To support our experimental data, we conducted a series of molecular dynamics simulations, exploring the interactions between the ABH2 mutant forms, bearing corresponding substitutions and DNA substrates, and harboring various types of methylated bases, specifically N¹-methyladenine or N³-methylcytosine. The comparative studies revealed compelling data indicating that alterations in most of the studied amino acid residues significantly influence both the binding affinity of the enzyme for DNA and its catalytic efficiency. Intriguingly, the findings suggest that the mutations impact the catalytic activity of ABH2 to a greater extent than its ability to associate with DNA strands. Collectively, these results show how changes to the active site affect molecular dynamics and reaction kinetics, improving our understanding of the substrate recognition process in this pivotal enzyme. Full article

This study discloses the noncovalent immobilization of a bienzyme cascade composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto magnetically responsive polyamide microparticles (PA MPs). Porous PA6, PA4, and PA12 MPs containing iron fillers were synthesized via activated anionic ring-opening polymerization in suspension, alongside neat PA6 MPs used as a reference. Four hybrid catalytic systems (GOx/HRP@PA) were prepared through sequential adsorption of HRP and GOx onto the various PA MP supports. The initial morphologies of the supports and the hybrid biocatalysts were characterized by SEM, followed by evaluation of the catalytic performance using a two-step glucose oxidation cascade process. Among all systems, the GOx/HRP@PA4-Fe complex exhibited the highest activity, being approximately 1.5 times greater than the native enzyme dyad, followed by the PA6-supported system with slightly inferior performance. All systems obeyed Michaelis–Menten kinetics, with the immobilized cascades displaying higher Kₘ and Vₘₐₓ values than the non-immobilized enzyme pair while maintaining comparable catalytic efficiencies, CE (CE = k_cat/Kₘ). Subsequently, the immobilized and native enzyme systems were employed for the polymerization of aniline. According to UV–VIS, complete monomer conversion was achieved within 24 h for selected catalysts, and FTIR analysis confirmed the formation of polyaniline in the emeraldine base form without the use of template molecules. These findings highlight the potential of Fe-containing polyamide microparticles as efficient supports for the sustainable, enzyme-mediated synthesis of intrinsically conductive aromatic polymers. Full article

Lactate dehydrogenase (LDH) catalyzes the reversible interconversion of pyruvate and lactate, coupled with the redox cycling of NADH and NAD⁺. While LDHA has been extensively studied as a therapeutic target, particularly in cancer, due to its role in the Warburg effect, LDHB remains underexplored, despite its involvement in the metabolic reprogramming of specific cancer types, including breast and lung cancers. Most known LDH inhibitors are designed against the LDHA isoform and act competitively at the active site. In contrast, LDHB exhibits distinct kinetic properties, substrate preferences, and structural features, warranting isoform-specific screening strategies. In this study, 115 natural compounds previously reported as LDHA inhibitors were systematically evaluated for LDHB inhibition using an integrated in silico and in vitro approach. Virtual screening identified 16 lead phytochemicals, among which luteolin and quercetin exhibited uncompetitive inhibition of LDHB, as demonstrated by enzyme kinetic assays. These findings were strongly supported by molecular docking analyses, which revealed that both compounds bind at an allosteric site located at the dimer interface, closely resembling the binding mode of the established LDHB uncompetitive inhibitor AXKO-0046. In contrast, comparative docking against LDHA confirmed their active-site binding and competitive inhibition, underscoring their isoform-specific behavior. Our findings highlight the necessity of assay conditions tailored to LDHB’s physiological role and demonstrate the application of a previously validated colorimetric assay for high-throughput screening. This work lays the foundation for the rational design of selective LDHB inhibitors from natural product libraries. Full article

The non-monotonic behavior of amperometric enzyme-based biosensors under uncompetitive and noncompetitive (mixed) substrate inhibition is investigated computationally using a two-compartment model consisting of an enzyme layer and an outer diffusion layer. The model is based on a system of reaction–diffusion equations that includes a nonlinear term associated with non-Michaelis–Menten kinetics of the enzymatic reaction and accounts for the partitioning between layers. In addition to the known effect of substrate inhibition, where the maximum biosensor current differs from the steady-state output, it has been determined that external diffusion limitations can also cause the appearance of a local minimum in the current. At substrate concentrations greater than both the Michaelis–Menten constant and the uncompetitive substrate inhibition constant, and in the presence of external diffusion limitation, the transient response of the biosensor, after immersion in the substrate solution, may follow a five-phase pattern depending on the model parameter values: it starts from zero, reaches a global or local maximum, decreases to a local minimum, increases again, and finally decreases to a steady intermediate value. The biosensor performance is analyzed numerically using the finite difference method. Full article

Acid phosphatase (ACPase) is an essential industrial enzyme, but its production via recombinant bacterial fermentation is often limited by insufficient dissolved oxygen control. This study optimized the aerobic fermentation of the ACPase-producing recombinant bacterium Bacillus subtilis 168/pMA5-Acp by refining the bioreactor’s aerodynamic structure using computational fluid dynamics (CFD) simulations. This was combined with fermentation kinetics modeling to achieve precise process control. First, the gas distributor structure of the 5 L bioreactor was optimized using CFD simulation results. Optimal mass transfer conditions were identified through comprehensive analysis of K_La in different reactor regions (aeration ratio: 1.142 VVm, K_La = 264.2 h⁻¹). The simulation results showed that the optimized oxygen transfer efficiency increased 2.49 fold compared to the prototype. Second, the process control issue was addressed by developing a BP (backpropagation) neural network model to predict K_La under alternative media conditions. The prediction error was less than 5%, and the model was combined with the logistic equation to construct the bacterial growth kinetic model (R² > 0.99). The experiments demonstrated that using the optimized reactor with a molasses–urea medium (molasses 7.5 g/L; urea 15 g/L; K₂HPO₄ 1.2 g/L; MgSO₄·7H₂O 0.25 g/L) reduced production costs while maintaining enzyme activity (215.99 U/mL) and biomass (OD₆₀₀ = 101.67) by 90.03%. This study provides an efficient and cost-effective process solution for the industrial production of ACPase and a theoretical foundation for bioreactor design and scale-up. Full article

The emergence of antifungal-resistant Aspergillus fumigatus (A. fumigatus) became a serious public health concern, underscoring the need for new effective antifungal agents. Here, we present a strategy based on the in situ generation of radical species that are toxic to the pathogen. The synthesis of an alkoxyamine linked to a peptide substrate recognized by A. fumigatus-secreted dipeptidyl peptidase is described. Kinetic experiments show a stable prodrug prior to enzymatic activation. Ensuing peptide cleavage and spontaneous homolysis resulted in the generation of a stable nitroxide and a reactive alkyl radical moiety. Next, the exposure of A. fumigatus spores to the prodrug lead to pathogen growth inhibition in a compound concentration-dependent fashion (e.g., 42% inhibition at 10 µg/L). Importantly, the designed alkoxyamine inhibited not only the growth of a clinical voriconazole-susceptible A. fumigatus strain, but also the growth of a strain resistant to this azole. To determine the antifungal importance of the reactive alkyl radical, its substitution with a non-radical structure did not prevent A. fumigatus growth. Furthermore, the introduction of succinic group in the peptide substrate resulted in the loss of alkoxyamine antifungal properties. Our work reports a novel chemical strategy for antifungal therapy against A. fumigatus based on the pathogen enzyme-mediated generation of toxic radicals. Significantly, these findings are timely since they could overcome the emerged resistance to conventional drugs that are known to target defined pathogen biologic mechanisms such as ergosterol synthesis. Full article

This study explores the identification, characterization, and biological evaluation of angiotensin I-converting enzyme (ACE)-inhibitory peptides derived from enzymatic hydrolysates of crucian carp swim bladders. Following sequential purification by size-exclusion and reversed-phase chromatography, two bioactive peptides—Hyp-Gly-Ala-Arg (Hyp-GAR) and Gly-Ala-Hyp-Gly-Ala-Arg (GA-Hyp-GAR)—were identified using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap tandem mass spectrometry. The synthetic peptides demonstrated potent ACE-inhibitory activity in vitro, with IC₅₀ values of 12.2 μM (Hyp-GAR) and 4.00 μM (GA-Hyp-GAR). Molecular docking and enzyme kinetics confirmed competitive inhibition through key interactions with ACE active site residues and zinc coordination. In vivo antihypertensive activity was evaluated in spontaneously hypertensive rats, revealing that GA-Hyp-GAR significantly reduced systolic blood pressure in a dose-dependent manner. At a dose of 36 mg/kg, GA-Hyp-GAR reduced systolic blood pressure by 60 mmHg—an effect comparable in magnitude and timing to that of captopril. Mechanistically, GA-Hyp-GAR modulated levels of angiotensin II, bradykinin, endothelial nitric oxide synthase, and nitric oxide. A 90-day subchronic oral toxicity study in mice indicated no significant hematological, biochemical, or histopathological alterations, supporting the peptide’s safety profile. These findings suggest that GA-Hyp-GAR is a promising natural ACE inhibitor with potential application in functional foods or as a nutraceutical for hypertension management. Full article

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines and plant alkaloids, including medicinal tropane alkaloids (TAs). Due to its key role, ODC has been utilized as an effective molecular tool in metabolic engineering. However, to date, only a limited number of plant ODCs have been characterized. Among the reported ODCs, Erythroxylum coca ODC (EcODC) exclusively has ODC activity, while Nicotiana glutinosa ODC (NgODC) exhibits dual ODC and lysine decarboxylase (LDC) activities. The potential LDC activity of ODCs from TA-producing plants remains unknown. Here, we characterized AlODC and DsODC from Anisodus luridus and Datura stramonium, along with two previously reported ODCs from Atropa belladonna (AbODC) and Hyoscyamus niger (HnODC), in Escherichia coli to investigate their enzyme kinetics and substrate specificity. Enzymatic assays revealed that both AlODC and DsODC catalyzed the conversion of ornithine to putrescine, confirming their ODC activity, with AlODC exhibiting a higher catalytic efficiency, comparable to established ODCs. Furthermore, all four ODCs also displayed LDC activity, albeit at significantly lower efficiency (<1% of ODC activity). This study provides a comprehensive analysis of the enzyme kinetics of ODCs from TA-producing plants, identifying promising candidate genes for metabolic engineering for the biomanufacturing of putrescine-derived alkaloids. Moreover, this is the first report of LDC activity in ODCs from Solanaceae TA-producing plants, shedding light on the evolutionary relationship between ODC and LDC. Full article

Fractional calculus plays a central role in modeling memory-dependent processes and complex dynamics across various fields, including control theory, fluid mechanics, and bioengineering. This study introduces an efficient and stable fractional-order iterative method based on the Caputo derivative for solving nonlinear equations. By employing a Taylor series expansion, a local convergence analysis shows that for $\gamma \in (0,1]$, the method achieves a convergence order of $2\gamma +1$. To address challenges related to memory effects and instability in existing approaches, the proposed scheme incorporates parameter optimization through chaos and bifurcation analysis. Dynamical plane analysis reveals that parameter values within chaotic regimes lead to divergence, while those in stable regions converge uniformly. The method’s performance is evaluated using a set of nonlinear models drawn from biomedical engineering, including enzyme kinetics with inhibition, extended glucose–insulin regulation, drug dose–responses, and lung volume–pressure dynamics. Comparative results demonstrate that the proposed approach outperforms existing methods in terms of iteration count, residual error, CPU time, convergence order, fractal behavior, and memory efficiency. These findings underscore the method’s applicability to complex systems characterized by nonlinearity and memory effects in scientific and engineering contexts. Full article

The venom of Nemopilema nomurai jellyfish represents a promising source of bioactive compounds with potential pharmacological applications. In our previous work, we identified two novel angiotensin-converting enzyme (ACE)-inhibitory peptides—IVGRPLANG (896.48 Da) and IGDEPRHQYL (1227.65 Da)—isolated from N. nomurai venom hydrolysates via papain digestion. In this study, we conducted a detailed biochemical and computational characterization of these peptides. The IC₅₀ values were determined to be 23.81 µM for IVGRPLANG and 5.68 µM for IGDEPRHQYL. Kinetic analysis using Lineweaver–Burk plots revealed that both peptides act as competitive ACE inhibitors, with calculated inhibition constants (K_i) of 51.38 µM and 5.45 µM, respectively. To assess the structural stability of the ACE–peptide complexes, molecular dynamics simulations were performed. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analyses provided insights into complex stability, while interaction fraction analysis elucidated key bond types and residue–ligand contacts involved in binding. Furthermore, a network pharmacology approach was employed to predict therapeutic targets within the renin–angiotensin–aldosterone system (RAAS). Eleven target proteins were identified: IVGRPLANG was associated with REN, ACE, CTSB, CTSS, and AGTR2; IGDEPRHQYL was linked to REN, AGT, AGTR1, AGTR2, KNG1, and BDKR2. Molecular docking analyses using HADDOCK software (version 2.4) were conducted for all targets to evaluate binding affinities, providing further insight into the peptides’ therapeutic potential. Full article

The aromatic C₁₃ apocarotenoid β-ionone is a high-value natural-flavor and -fragrance compound derived from the oxidative cleavage of carotenoids. Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, particularly β-ionone. In this study, we report the identification, cloning, and functional characterization of two CCD1 homologs: OeCCD1 from Olea europaea and InCCD1 from Ipomoea nil. These two species, which, respectively, represent a woody perennial and a herbaceous annual, were selected to explore the potential functional divergence of CCD1 enzymes across different plant growth forms. These CCD1 genes were synthesized using codon optimization for Escherichia coli expression, followed by heterologous expression and purification using a GST-fusion system. In vitro assays confirmed that both enzymes cleave β-carotene at the 9,10 (9′,10′) double bond to yield β-ionone, but only OeCCD1 exhibits detectable activity on zeaxanthin; InCCD1 shows no in vitro cleavage of zeaxanthin. Kinetic characterization using β-apo-8′-carotenal as substrate revealed, for OeCCD1, a K_m of 0.82 mM, V_max of 2.30 U/mg (k_cat = 3.35 s⁻¹), and k_cat/K_m of 4.09 mM⁻¹·s⁻¹, whereas InCCD1 displayed K_m = 0.69 mM, V_max = 1.22 U/mg (k_cat = 1.82 s⁻¹), and k_cat/K_m = 2.64 mM⁻¹·s⁻¹. The optimization of expression parameters, as well as the systematic evaluation of temperature, pH, solvent, and metal ion effects, provided further insights into the stability and functional diversity within the plant CCD1 family. Overall, these findings offer promising enzymatic tools for the sustainable production of β-ionone and related apocarotenoids in engineered microbial cell factories. Full article

Obesity is a chronic epidemic caused by abnormal fat metabolism. As a key digestive enzyme, pancreatic lipase (PL) is an important target for regulating fat metabolism. The inhibitory potential of 5,10,15,20-Tetrakis (4-aminophenyl) porphyrin (TAPP), 5,10,15,20-Tetrakis (4-hydroxyphenyl) porphyrin (THPP), meso-Tetra (4-carboxyphenyl) porphine (TCPP), Cu (II) meso-Tetra (4-carboxyphenyl) porphine (Cu-TCPP) on PL was studied by enzymatic kinetics, multi-spectral, and molecular simulation technology. THPP, TCPP, TAPP, and Cu-TCPP all had good PL inhibitory activity (IC₅₀ range: 97.49–248.70 μM) and were uncompetitive inhibitors. The order of inhibitory ability was: THPP > TCPP > TAPP > Cu-TCPP. The fluorescence quenching mechanism of THPP to PL was a mixed quenching dominated by static quenching, while TCPP, TAPP, and Cu-TCPP were static quenching. The binding of THPP, TCPP and TAPP to PL was mainly driven by hydrogen bonds and van der Waals forces, while Cu-TCPP was mainly driven by a hydrophobic interaction. Four porphyrin compounds changed the conformation of PL, affected the microenvironment of Tyr and Trp residues, and induced changes in the secondary structure of PL, thereby reducing the stability and catalytic activity of PL. Hydrogen bonds played an important role in the binding stability of THPP, TCPP, TAPP, and PL. Full article

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α₂β₂γ₂ heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α₂β₂γ₂ complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme. Full article

Plastic waste accumulation presents critical environmental challenges demanding innovative circular economy solutions. This study developed a comprehensive machine learning framework to systematically identify optimal enzyme combinations for polyester depolymerization. We integrated kinetic parameters from the BRENDA database with sequence-derived features and network topology metrics to train ensemble classifiers predicting enzyme-substrate relationships. A multi-objective optimization algorithm evaluated enzyme combinations across four criteria: prediction confidence, substrate coverage, operational compatibility, and functional diversity. The ensemble classifier achieved 86.3% accuracy across six polymer families, significantly outperforming individual models. Network analysis revealed a modular organization with hub enzymes exhibiting broad substrate specificity. Multi-objective optimization identified 156 Pareto-optimal enzyme combinations, with top-ranked pairs achieving composite scores exceeding 0.89. The Cutinase–PETase combination demonstrated exceptional complementarity (score: $0.875\pm 0.008$), combining complete substrate coverage with high catalytic efficiency. Validation against experimental benchmarks confirmed enhanced depolymerization rates for recommended enzyme cocktails. This framework provides a systematic approach for enzyme prioritization in plastic valorization, advancing biological recycling technologies through data-driven biocatalyst selection while identifying key economic barriers requiring technological innovation. Full article

The fibrin matrix of thrombi is intertwined with neutrophil extracellular traps (NETs) containing histones that render resistance to fibrinolysis. During NET formation, histones are citrullinated. Our study addresses the question of whether citrullination modifies the fibrin-stabilizing effects of histones. We studied the structure and viscoelastic properties of fibrin formed in the presence of native or citrullinated H1 and core histones by scanning electron microscopy, clot permeation, and oscillation rheometry. The kinetics of fibrin formation and its dissolution were followed by turbidimetry and thromboelastometry. Co-polymerizing H1 with fibrin enhanced the mechanical strength of the clots, thickened the fibrin fibers, and enlarged the gel pores. In contrast, the addition of core histones resulted in a reduction in the fiber diameter, and the pores were only slightly larger, whereas the mechanical stability was not modified. Plasmin-mediated fibrinogen degradation was delayed by native and citrullinated core histones, but not by H1, and the action of des-kringle1-4-plasmin was not affected. Plasmin-mediated fibrinolysis was inhibited by native and citrullinated core histones, and this effect was moderated when the kringle domains of plasmin were blocked or deleted. These findings suggest that in NET-containing thrombi that are rich in core histones, alternative fibrinolytic enzymes lacking kringle domains are more efficient lytic agents than the classic plasmin-dependent fibrinolysis. Full article