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Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2-deoxyguanosine) are major products of 2-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer-dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to 2-deoxyguanosine. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G, or U. Fitting of reaction data indicates that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast- or slow-reacting base pairs vary less than twofold as a function of the nucleotide opposite the lesion. Promoter-dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side by side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

Enzymes accelerate the rates of biomolecular reactions by many orders of magnitude compared to bulk solution, and it is widely understood that this catalytic effect arises from a combination of polar pre-organization and electrostatic transition state stabilization. A number of recent reports have also implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity. However, complications arising from spatially-distributed disorder, the occurrence of multiple substrate binding modes, and the influence of hydration dynamics on solvent-exposed active sites still confound many experimental studies. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the promiscuous Pyrococcus horikoshii ene-reductase (PhENR) active site in two binding configurations mimicking proposed "inactive" and "reactive" Michaelis complexes. Spectral diffusion measurements of aryl-nitrile substrate analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5 ps) motions. Fermi resonant aryl-azide analogs that sense interactions of coupled oscillators are described. Lineshape and quantum beat analyses of these probes reveal characteristics that correlate with aryl-nitrile frequency fluctuation correlation functions parameters, demonstrating that this anisotropy is an intrinsic property of the water-exposed active site, where countervailing gradients of fast dynamics and disorder in the reactant ground state are maintained near the hydration interface. Our results suggest several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR. This study also highlights a strategy to detect perturbations to vibrational modes outside the transparent window of the mid-IR spectrum, which may be extended to other macromolecular systems. [ABSTRACT FROM AUTHOR]

Classical theories of enzyme inhibition kinetics predict a monotonic decrease in the mean catalytic activity with the increase in inhibitor concentration. The steady-state result, derived from deterministic mass action kinetics, ignores molecular noise in enzyme-inhibition mechanisms. Here, we present a stochastic generalization of enzyme inhibition kinetics to mesoscopic enzyme concentrations by systematically accounting for molecular noise in competitive and uncompetitive mechanisms of enzyme inhibition. Our work reveals an activator-inhibitor duality as a non-classical effect in the transient regime in which inhibitors tend to enhance enzymatic activity. We introduce statistical measures that quantify this counterintuitive response through the stochastic analog of the Lineweaver–Burk plot that shows a merging of the inhibitor-dependent velocity with the Michaelis–Menten velocity. The statistical measures of mean and temporal fluctuations - fractional enzyme activity and waiting time correlations - show a non-monotonic rise with the increase in inhibitors before subsiding to their baseline value. The inhibitor and substrate dependence of the fractional enzyme activity yields kinetic phase diagrams for non-classical activator-inhibitor duality. Our work links this duality to a molecular memory effect in the transient regime, arising from positive correlations between consecutive product turnover times. The vanishing of memory in the steady state recovers all the classical results. [ABSTRACT FROM AUTHOR]

We present an analysis of high-resolution quasi-elastic neutron scattering spectra of phosphoglycerate kinase which elucidates the influence of the enzymatic activity on the dynamics of the protein. We show that in the active state the inter-domain motions are amplified and the intra-domain asymptotic power-law relaxation ∝t−α is accelerated, with a reduced coefficient α. Employing an energy landscape picture of protein dynamics, this observation can be translated into a widening of the distribution of energy barriers separating conformational substates of the protein. [ABSTRACT FROM AUTHOR]

In this study, we aimed to investigate the inhibitory mechanisms of β‐glucuronidase by flavonoids derived from Alhagi graecorum through both experimental and computational approaches. The activity of β‐glucuronidase was assessed using an in vitro enzyme inhibition assay, where myricetin and chrysoeriol were identified as potent inhibitors based on their low IC50 values. Kinetic studies were conducted to determine the inhibition type, revealing that both compounds exhibit noncompetitive inhibition of β‐glucuronidase‐catalyzed hydrolysis of PNPG. Molecular docking was employed to explore the binding affinities of the flavonoids, showing that myricetin formed the highest number of polar interactions with the enzyme. Additionally, molecular dynamics (MD) simulations were performed to evaluate the stability of the enzyme‐inhibitor complexes, demonstrating consistent trajectory behavior for both compounds, with significant energy stabilization. Interaction energy analyses highlighted the dominant role of electrostatic forces in myricetin′s inhibition mechanism, while Van der Waals forces were more prominent for chrysoeriol. The MM/PBSA method was used to calculate the binding free energies, with myricetin and chrysoeriol exhibiting the lowest values. Potential energy landscape analysis further revealed that β‐glucuronidase adopts a more closed conformation when bound to these inhibitors, limiting substrate access. These findings suggest that flavonoids from Alhagi graecorum hold promise for clinical applications, particularly in managing drug‐induced enteropathy. [ABSTRACT FROM AUTHOR]

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The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca2+ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca2+-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca2+/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca2+/cAMP oscillation reaches Ca2+ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca2+-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca2+/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca2+/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca2+/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca2+ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers. Author summary: Cyclic adenosine monophosphate (cAMP) and Ca2+ are key molecules that relay signals from the cell membrane to downstream molecules. The temporal and spatial distribution of cAMP and Ca2+ within the cell can be regulated by clusters formed by specific molecules. Previous studies have shown that nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates Ca2+-cAMP oscillations from non-zero to zero phase difference, i.e., from out-of-phase to in-phase. By developing a computational model of the Ca2+/cAMP pathway, we found that the non-zero phase difference is not an arbitrary value: it makes Ca2+ reach trough and cAMP reach peak simultaneously. We defined this out-of-phase behavior as inversely out-of-phase, and revealed that this behavior and in-phase behavior can be explained by an incoherent feedforward loop. Biophysical properties of cells and nanodomain distributions do not affect the existence of in-phase or inversely out-of-phase behaviors, but may affect the time delay for the inversely out-of-phase behavior. Furthermore, AKAP/AC nanodomains from a Turing pattern or experiments show consistent simulation results with the incoherent feedforward loop. This study improves our understanding of underlying mechanisms of Ca2+/cAMP oscillation, shedding light on controlling Ca2+ and cAMP through nanodomains. [ABSTRACT FROM AUTHOR]

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2'-deoxyuridine 5'-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2'-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release. [ABSTRACT FROM AUTHOR]

Designing and developing small organic molecules for use as urease inhibitors is challenging due to the need for ecosystem sustainability and the requirement to prevent health risks related to the human stomach and urinary tract. Moreover, imaging analysis is widely utilized for tracking infections in intracellular and in vivo systems, which requires drug molecules with emissive potential, specifically in the low-energy region. This study comprises the synthesis of a Schiff base ligand and its selected transition metals to evaluate their UV/fluorescence properties, inhibitory activity against urease, and molecular docking. Screening of the symmetrical cage-like ligand and its metal complexes with various eco-friendly transition metals revealed significant urease inhibition potential. The IC50 value of the ligand for urease inhibition was 21.80 ± 1.88 µM, comparable to that of thiourea. Notably, upon coordination with transition metals, the ligand–nickel and ligand–copper complexes exhibited even greater potency than the reference compound, with IC50 values of 11.8 ± 1.14 and 9.31 ± 1.31 µM, respectively. The ligand–cobalt complex exhibited an enzyme inhibitory potential comparable with thiourea, while the zinc and iron complexes demonstrated the least activity, which might be due to weaker interactions with the investigated protein. Meanwhile, all the metal complexes demonstrated a pronounced optical response, which could be utilized for fluorescence-guided targeted drug delivery applications in the future. Molecular docking analysis and IC50 values from in vitro urease inhibition screening showed a trend of increasing activity from compounds 7d to 7c to 7b. Enzyme kinetics studies using the Lineweaver–Burk plot indicated mixed-type inhibition against 7c and non-competitive inhibition against 7d. [ABSTRACT FROM AUTHOR]

Copper-containing diamine oxidases are ubiquitous enzymes that participate in many important biological processes. These processes include the regulation of cell growth and division, programmed cell death, and responses to environmental stressors. Natural substrates include, for example, putrescine, spermidine, and histamine. Enzymatic activity is typically assayed using spectrophotometric, electrochemical, or fluorometric methods. The aim of this study was to develop a method for measuring activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based on the intensity ratio of product to product-plus-substrate signals in the reaction mixtures. For this purpose, an enzyme purified to homogeneity from pea (Pisum sativum) seedlings was used. The method employed α-cyano-4-hydroxycinnamic acid as a matrix with the addition of cetrimonium bromide. Product signal intensities with pure compounds were evaluated in the presence of equal substrate amounts to determine intensity correction factors for data processing calculations. The kinetic parameters kcat and Km for the oxidative deamination of selected substrates were determined. These results were compared to parallel measurements using an established spectrophotometric method, which involved a coupled reaction of horseradish peroxidase and guaiacol, and were discussed in the context of data from the literature and the BRENDA database. It was found that the method provides accurate results that are well comparable with parallel spectrophotometry. This method offers advantages such as low sample consumption, rapid serial measurements, and potential applicability in assays where colored substances interfere with spectrophotometry. [ABSTRACT FROM AUTHOR]

A series of thiadiazole derivatives were designed and synthesized as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) for the treatment of Alzheimer's disease (AD). Furthermore, the compounds were assayed for their inhibitory activity to AChE and BuChE in vitro, and the results indicated that most of the compounds had moderate inhibitory activity to AChE and BuChE. Among them, compound 11 u showed the best inhibitory activity against AChE and BuChE (eeAChE, IC50=2.73 μM; eqBuChE, IC50=0.65 μM), which showed approximately 2‐fold more activity against AChE enzyme than galantamine and approximately 28‐fold more activity against BuChE enzyme than galantamine. In addition, molecular docking studies have shown that compound 11 u could bind to the catalytic active site (CAS) and peripheral anion site (PAS) of AChE and BuChE. Among them, compound 11 u combined with BuChE and exhibited a mixed inhibition pattern consistent with enzyme kinetics studies. The interaction's stability of 11 u‐AChE/BuChE were also assessed using a conventional atomistic 100 ns dynamics simulation study, which revealed the conformational stability of representative compound 11 u in the cavity of the AChE/BuChE. Moreover, the molecular properties of all compounds were predicted by online through the SwissADME, and the best active compound 11 u matched the properties of most orally administered drugs. All these suggested that 11 u could be considered as a lead compound for the development of drugs for AD. [ABSTRACT FROM AUTHOR]

6-Gingerol is a major phenolic compound within ginger (Zingiber officinale), often used in healthcare; however, its lower bioavailability is partly due to its poor solubility. Four bacterial glycosyltransferases (GTs) were tested to glycosylate 6-gingerol into soluble gingerol glucosides. BsUGT489 was a suitable GT to biotransform 6-gingerol into five significant products, which could be identified via nucleic magnetic resonance and mass spectrometry as 6-gingerol-4′,5-O-β-diglucoside (1), 6-gingerol-4′-O-β-glucoside (2), 6-gingerol-5-O-β-glucoside (3), 6-shogaol-4′-O-β-glucoside (4), and 6-shogaol (5). The enzyme kinetics of BsUGT489 showed substrate inhibition toward 6-gingerol for producing two glucosides. The kinetic parameters were determined as KM (110 μM), kcat (862 min-1), and KI (571 μM) for the production of 6-gingerol-4′-O-β-glucoside (2) and KM (104 μM), kcat (889 min-1), and KI (545 μM) for the production of 6-gingerol-5-O-β-glucoside (3). The aqueous solubility of the three 6-gingerol glucosides, compound (1) to (3), was greatly improved. However, 6-shogaol-4′-O-β-glucoside (4) was found to be a product biotransformed from 6-shogaol (5). This study first confirmed that the glucose moiety at the C-5 position of both 6-gingerol-4′,5-O-β-diglucoside (1) and 6-gingerol-5-O-β-glucoside (3) caused spontaneous deglucosylation through β-elimination to form 6-shogaol-4′-O-β-glucoside (4) and 6-shogaol (5), respectively. Moreover, the GTs could glycosylate 6-shogaol to form 6-shogaol-4′-O-β-glucoside (4). The assays showed 6-shogaol-4′-O-β-glucoside (4) had higher anti-inflammatory activity (IC50 value of 10.3 ± 0.2 μM) than 6-gingerol. The 6-gingerol-5-O-β-glucoside (3) possessed 346-fold higher solubility than 6-shogaol, in which the highly soluble glucoside is a potential prodrug of 6-shogaol via spontaneous deglucosylation. This unusual deglucosylation plays a vital role in influencing the anti-inflammatory activity. IMPORTANCE Both 6-gingerols and 6-shogaol possess multiple bioactivities. However, their poor solubility limits their application. The present study used bacterial GTs to catalyze the glycosylation of 6-gingerol, and the resulting gingerol glycosides were found to be new compounds with improved solubility and anti-inflammatory activity. In addition, two of the 6-gingerol glucosides were found to undergo spontaneous deglucosylation to form 6-shogaol or 6-shogaol glucosides. The unique spontaneous deglucosylation property of the new 6-gingerol glucosides makes them a good candidate for the prodrug of 6-shogaol. [ABSTRACT FROM AUTHOR]

To develop the drugs as a second line of preventing a serious form of illness, blocking the interaction between a receptor‐binding domain (RBD) in the SARS‐CoV‐2 S‐protein (spike protein) with human ACE2 (Angiotensin converting enzyme 2) can potentially prevent SARS‐CoV‐2 S‐protein from interacting with host cells. In this research, 20 drug compounds are examined using docking to identify potential drugs that can bind at the common level of the RBD‐ACE2 complex and compared the results with two standard drugs offered (Favipiravir, Arbidol). Among 20 drugs, Ozanimod and Prazosin are selected as the best drug compounds by reviewing the docking scores and drug interaction with the active position of RBD‐ACE2. The results of molecular dynamics simulation showed that Ozanimod with binding energy of −14.24 kcal mol−1 has a higher binding capability than Prazosin with binding energy of −9.55 kcal mol−1 to block the interaction between spike protein RBD and human ACE2 enzyme. Ozanimod effectively binds to the S‐protein RBD and inhibits residues critical to the spike and ACE2 protein interaction. This drug compound is expected to be a potentially effective inhibitor of the interaction between the S‐ RBD and the human ACE2 enzyme. [ABSTRACT FROM AUTHOR]

Purpose: This study explores the immobilisation of enzymes within porous catalysts of various geometries, including spheres, cylinders and flat pellets. The objective is to understand the irreversible Michaelis-Menten kinetic process within immobilised enzymes through advanced mathematical modelling. Design/methodology/approach: Mathematical models were developed based on reaction-diffusion equations incorporating nonlinear variables associated with Michaelis-Menten kinetics. This research introduces fractional derivatives to investigate enzyme reaction kinetics, addressing a significant gap in the existing literature. A novel approximation method, based on the independent polynomials of the complete bipartite graph, is employed to explore solutions for substrate concentration and effectiveness factor across a spectrum of parameter values. The analytical solutions generated through the bipartite polynomial approximation method (BPAM) are rigorously tested against established methods, including the Bernoulli wavelet method (BWM), Taylor series method (TSM), Adomian decomposition method (ADM) and fourth-order Runge-Kutta method (RKM). Findings: The study identifies two main findings. Firstly, the behaviour of dimensionless substrate concentration with distance is analysed for planar, cylindrical and spherical catalysts using both integer and fractional order Michaelis-Menten modelling. Secondly, the research investigates the variability of the dimensionless effectiveness factor with the Thiele modulus. Research limitations/implications: The study primarily focuses on mathematical modelling and theoretical analysis, with limited experimental validation. Future research should involve more extensive experimental verification to corroborate the findings. Additionally, the study assumes ideal conditions and uniform catalyst properties, which may not fully reflect real-world complexities. Incorporating factors such as mass transfer limitations, non-uniform catalyst structures and enzyme deactivation kinetics could enhance the model's accuracy and broaden its applicability. Furthermore, extending the analysis to include multi-enzyme systems and complex reaction networks would provide a more comprehensive understanding of biocatalytic processes. Practical implications: The validated bipartite polynomial approximation method presents a practical tool for optimizing enzyme reactor design and operation in industrial settings. By accurately predicting substrate concentration and effectiveness factor, this approach enables efficient utilization of immobilised enzymes within porous catalysts. Implementation of these findings can lead to enhanced process efficiency, reduced operating costs and improved product yields in various biocatalytic applications such as pharmaceuticals, food processing and biofuel production. Additionally, this research fosters innovation in enzyme immobilisation techniques, offering practical insights for engineers and researchers striving to develop sustainable and economically viable bioprocesses. Social implications: The advancement of enzyme immobilisation techniques holds promise for addressing societal challenges such as sustainable production, environmental protection and healthcare. By enabling more efficient biocatalytic processes, this research contributes to reducing industrial waste, minimizing energy consumption and enhancing access to pharmaceuticals and bio-based products. Moreover, the development of eco-friendly manufacturing practices through biocatalysis aligns with global efforts towards sustainability and mitigating climate change. The widespread adoption of these technologies can foster a more environmentally conscious society while stimulating economic growth and innovation in biotechnology and related industries. Originality/value: This study offers a pioneering approximation method using the independent polynomials of the complete bipartite graph to investigate enzyme reaction kinetics. The comprehensive validation of this method through comparison with established solution techniques ensures its reliability and accuracy. The findings hold promise for advancing the field of biocatalysts and provide valuable insights for designing efficient enzyme reactors. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid 'P' within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest kcat of 0.95 s−1, followed by rDHFR with 0.14 s−1, while eDHFR displayed the lowest kcat of 0.09 s−1. Remarkably, the K65P variant induced a significant reduction in Km, resulting in a 1.8-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue. [ABSTRACT FROM AUTHOR]

Hormone receptor (HR)-positive breast cancer represents tumours that express estrogen and/or progesterone enzymes, which play a crucial role in the growth and proliferation of breast cancer cells. Aromatase inhibitors (AIs) are drugs that inhibit the enzyme aromatase, involved in the biosynthesis of estrogen and are used for the treatment of HR-positive breast cancer. The USFDA-approved nonsteroidal AIs contain 1,2,4-triazole heterocycle. So, in the present study, we screened seventy-eight 1,2,4-triazole analogues from different literature resources and generated a pharmacophore model. After several validation protocols, Hypo1 was selected as the best model and was used as a 3D query for screening 1,2,4-triazole analogues (15,583) obtained from the CHEMBL database. A dataset containing 320 ligands with estimated activity (IC50< 0.1 µM) was subjected to molecular docking against the aromatase enzyme (PDB ID: 3S79). Amongst the 320 ligands, 30 compounds exhibited better binding energy compared to the standard drug letrozole. These 30 hits were further considered for the design of two novel molecules which were initially assessed for their ADMET properties followed by pharmacophore mapping, docking and molecular dynamics simulation. The in silico findings suggest that both the designed molecules can be considered drug-like candidates for the treatment of breast cancer through aromatase inhibition. [ABSTRACT FROM AUTHOR]

Background and Purpose: Demand for new antidepressants has resulted in a re‐evaluation of the therapeutic potential of psychedelic drugs. Several tryptamines found in psilocybin‐containing "magic" mushrooms share chemical similarities with psilocybin. Early work suggests they may share biological targets. However, few studies have explored their pharmacological and behavioural effects. Experimental Approach: We compared baeocystin, norbaeocystin and aeruginascin with psilocybin to determine if they are metabolized by the same enzymes, similarly penetrate the blood–brain barrier, serve as ligands for similar receptors and modulate behaviour in rodents similarly. We also assessed the stability and optimal storage and handling conditions for each compound. Key Results: In vitro enzyme kinetics assays found that all compounds had nearly identical rates of dephosphorylation via alkaline phosphatase and metabolism by monoamine oxidase. Further, we found that only the dephosphorylated products of baeocystin and norbaeocystin crossed a blood–brain barrier mimetic to a similar degree as the dephosphorylated form of psilocybin, psilocin. The dephosphorylated form of norbaeocystin was found to activate the 5‐HT2A receptor with similar efficacy to psilocin and norpsilocin in in vitro cell imaging assays. Behaviourally, only psilocybin induced head twitch responses in rats, a marker of 5‐HT2A‐mediated psychedelic effects and hallucinogenic potential. However, like psilocybin, norbaeocystin improved outcomes in the forced swim test. All compounds caused minimal changes to metrics of renal and hepatic health, suggesting innocuous safety profiles. Conclusions and Implications: Collectively, this work suggests that other naturally occurring tryptamines, especially norbaeocystin, may share overlapping therapeutic potential with psilocybin, but without causing hallucinations. [ABSTRACT FROM AUTHOR]

• Total As, As fractions and phosphatase activity reached a significant level. • V max and V max / K m of phosphatase decreased with elevated As pollution. • V max and K m were significantly negatively correlated with soil pH. • Phosphatase is more sensitive to As contamination at the soil's pH. • V max / K m as an index of pollution degree in As field contaminated soil. The pollution and ecological risks posed by arsenic (As) entering the soil are the major environmental challenges faced by human beings. Soil phosphatase can serve as a useful indicator for assessing As contamination under specific soil pH conditions. However, the study of phosphatase kinetics in long-term field As-contaminated soil remains unclear, presenting a significant obstacle to the monitoring and evaluation of As pollution and toxicity. The purpose of this study was to determine phosphatase activity and explore enzyme kinetics in soils subjected to long-term field As contamination. Results revealed that the soil phosphatase activity varied among the tested soil samples, depending on the concentrations of As. The relationship between total As, As fractions and phosphatase activity was found to be significant through negative exponential function fitting. Kinetic parameters, including maximum reaction velocity (V max), Michaelis constant (K m) and catalytic efficiency (V max / K m), ranged from 3.14 × 10−2–53.88 × 10−2 mmol/(L·hr), 0.61–7.92 mmol/L, and 0.46 × 10−2–11.20 × 10−2 hr−1, respectively. V max and V max / K m of phosphatase decreased with increasing As pollution, while K m was less affected. Interestingly, V max / K m showed a significant negative correlation with all As fractions and total As. The ecological doses (ED 10) for the complete inhibition and partial inhibition models ranged from 0.22–70.33 mg/kg and 0.001–55.27 mg/kg, respectively, indicating that V max / K m can be used as an index for assessing As pollution in field-contaminated soil. This study demonstrated that the phosphatase kinetics parameters in the soil's pH system were better indicators than the optimal pH for evaluating the field ecotoxicity of As. [Display omitted] [ABSTRACT FROM AUTHOR]

Kinetic chemical reactions find applications across various fields. In industrial processes, they drive the production of essential materials like fertilizers and pharmaceuticals. In environmental science, they are crucial to understanding pollution dynamics. Additionally, in biochemistry, they underpin vital cellular processes, offering insights into disease mechanisms and drug development. In this work, we present a new advancement of a dynamical system for kinetically controlled chemical reactions and the dependency of its solution on the initial conditions using mathematical techniques for fractional orders. By utilizing this fixed-point approach, we can derive the existence and uniqueness theorem of the proposed model. We further show that the chemical kinetics of the fractional model are stable through the Hyers-Ulam stability condition. We finally run a numerical simulation to verify our conclusions. The manuscript concludes with demonstrative examples. [ABSTRACT FROM AUTHOR]

Beta-galactosidase (EC 3.2.1.23), also known as lactase, is an exoglycosidase enzyme responsible for hydrolyzing the β-glycosidic bond between galactose and its organic moiety. This study focused on extracting, purifying, characterizing and immobilizing β-galactosidase from tomato leaves. The enzyme was successfully extracted using a 0.2 M sodium phosphate buffer at pH 6. To evaluate its chemical properties, the effects of temperature, pH, enzyme concentration and substrate concentration on enzyme activity were examined. The optimal temperature for β-galactosidase activity was found to be 50°C and the enzyme demonstrated the highest solubility at pH 6. The kinetic parameters of the free enzyme were determined, revealing a Michaelis constant (Km) of 0.1 mM and a maximum reaction velocity (Vmax) of 48.78 ìmol/min. These findings provide valuable insights into the enzyme's efficiency and behaviour under various conditions, which are essential for optimizing its application in industrial processes. [ABSTRACT FROM AUTHOR]

An analytic solution is presented for the simultaneous substrate elimination problem that combines Michaelis-Menten (MM) consumption with an irreversible homo-dimerization process. The implicit solution involves logarithm and inverse tangent functions and perfectly agrees with the numerical solution of the differential equation. A solution is also presented for the generalized dynamical problem that simultaneously combines MM kinetics with first and second-order processes. The exact expressions for the half-life and the area under the curve are also presented for these problems. [ABSTRACT FROM AUTHOR]

Adaptation of the response to stimuli is a fundamental process for all organisms. Here, we show that the adaptation enzyme CheB methylesterase of Escherichia coli assembles to the ON state receptor array after exposure to the repellent l-isoleucine and dissociates from the array after adaptation is complete. The duration of increased CheB localization and the time of highly clockwise-biased flagellar rotation were similar and depended on the strength of the stimulus. The increase in CheB at the receptor array and the decrease in cytoplasmic CheB were both ~100 molecules, which represents 15 to 20% of the total cellular content of CheB. We confirmed that the main binding site for CheB in the ON state array is the P2 domain of phosphorylated CheA, with a second minor site being the carboxyl-terminal pentapeptide of the serine chemoreceptor. Thus, we have been able to quantify the regulation of the signal output of the receptor array by the intracellular dynamics of an adaptation enzyme. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a major cause of blindness that affects people over 60. While aging is the prominent factor in AMD, studies have reported a higher prevalence of AMD in women compared to age-matched men. Higher levels of the innate immune response's effector proteins complement factor B and factor I were also found in females compared to males in intermediate AMD. However, the mechanisms underlying these differences remain elusive. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolic pathways. Previously, we showed that Pgc-1α repression and high-fat diet induce drastic AMD-like phenotypes in mice. Our recent data revealed that Pgc-1α repression alone can also induce retinal pigment epithelium (RPE) and retinal dysfunction in mice, and its inhibition in vitro results in lipid droplet accumulation in human RPE. Whether sex is a contributing factor in these phenotypes remains to be elucidated. Using electroretinography, we demonstrate that sex could influence RPE function during aging independent of Pgc-1α in wild-type (WT) mice. We further show that Pgc-1α repression exacerbates RPE and retinal dysfunction in females compared to aged-match male mice. Gene expression analyses revealed that Pgc-1α differentially regulates genes related to antioxidant enzymes and mitochondrial dynamics in males and females. RPE flat mounts immunolabeled with TOMM20 and DRP1 indicated a sex-dependent role for Pgc-1α in regulating mitochondrial fission. Analyses of mitochondrial network morphology suggested sex-dependent effects of Pgc-1α repression on mitochondrial dynamics. Together, our study demonstrates that inhibition of Pgc-1α induces a sex-dependent decline in RPE and retinal function in mice. These observations on the sex-dependent regulation of RPE and retinal function could offer novel insights into targeted therapeutic approaches for age-related RPE and retinal degeneration. [ABSTRACT FROM AUTHOR]

Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are membraneless organelles that orchestrate various metabolic pathways in living cells. Understanding how these crowded structures regulate enzyme reactions remains yet challenging due to their dynamic and intricate nature. Coacervate microdroplets formed by associative liquid‐liquid phase separation of oppositely charged polyions have emerged as relevant condensate models to study enzyme catalysis. Enzyme reactions within these droplets show altered kinetics, influenced by factors such as enzyme and substrate partitioning, crowding, and interactions with coacervate components; it is often challenging to disentangle the contributions of each. Here, we investigate the peroxidase activity of a de novo enzyme within polysaccharide‐based coacervates. By comparing the reaction kinetics in buffer, in a suspension of coacervates and in the bulk coacervate phase collected after centrifugation of the droplets, we show that the coacervate phase significantly increases the enzyme catalytic efficiency. We demonstrate that the main origin of this enhanced activity lies in macromolecular crowding coupled to changes in the conformational dynamics of the enzyme within the coacervate environment. Altogether, these findings underline the crucial role of the coacervate matrix in enzyme catalysis, beyond simple partitioning effects. The observed boost in enzyme activity within the coacervate phase provides insights for designing biocatalytically active synthetic organelles. [ABSTRACT FROM AUTHOR]

A few computational studies indicated that some inhibitors of viral RNA–polymerase such as favipiravir could also inhibit cysteine proteases. The potential dual action of favipiravir as an inhibitor for both RNA–polymerase and proteases promises enhanced therapeutic efficacy of the drug against coronaviruses. To shed more light on this phenomenon, in view of the chemical structure of favipiravir and the recent advances in this field, six novel derivatives of 3-methyl quinoxaline were synthesized. In silico methods confirmed the abilities of these compounds to occupy the active-site clefts of both 3CLpro and PLpro with favorable interactions, while the ADMET evaluations predicted low toxicity and high bioavailability for them. Then, due to the high similarities between PLpro and papain, the inhibitory impacts of favipiravir and the synthesized quinoxalines on the kinetics of papain were thoroughly investigated. The outcome revealed that the potential dual-action of favipiravir-like compounds is a possibility that should be taken into account in designing more effective and safe drugs against coronavirus. [ABSTRACT FROM AUTHOR]

Loss-of-function Parkin mutations lead to early-onset of Parkinson’s disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity [ABSTRACT FROM AUTHOR]

The heterogeneous distribution of lipids and lipid-bound proteins in a plasma membrane has functional advantages. Herein, in a synthetic system, we demonstrate the assembly of three enzymes involved in cascade reactions, in response to the substrate of the first enzyme at the oil–water interface being stabilized by a Zn(II)-metallosurfactant. Then we show substrate-mediated catalytically-active cluster formation of the metallosurfactant in a binary mixture with another non-catalytic surfactant at the interface. The catalytic ability can be tuned by controlling clustering through the addition of phosphate ions. Overall this work demonstrates functionally diverse supramolecular nanoarchitectonics at the oil–water interface. [ABSTRACT FROM AUTHOR]

Organic carbon fixed in chloroplasts through the Calvin–Benson–Bassham Cycle can be diverted toward different metabolic fates, including cytoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis–gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis–gluconeogenesis, Enolase and bis-phosphoglycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high-latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic, and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway. Environmental and experimental methods reveal the importance of plastid-targeted Enolase and bis-phosphoglycerate mutase enzymes to diatoms, marine algae that contribute to global primary production. [ABSTRACT FROM AUTHOR]

Alginate is a polysaccharide consumed by humans in edible seaweed and different foods where it is applied as a texturizing hydrocolloid or in encapsulations of drugs and probiotics. While gut bacteria are found to utilize and ferment alginate to health-beneficial short-chain fatty acids, knowledge on the details of the molecular reactions is sparse. Alginates are composed of mannuronic acid (M) and its C-5 epimer guluronic acid (G). An alginate-related polysaccharide utilization locus (PUL) has been identified in the gut bacterium Bacteroides eggerthii DSM 20697. The PUL encodes two polysaccharide lyases (PLs) from the PL6 (BePL6) and PL17 (BePL17) families as well as a KdgF-like metalloprotein (BeKdgF) known to catalyze ring-opening of 4,5-unsaturated monouronates yielding 4-deoxy-L-erythro-5-hexoseulose uronate (DEH). B. eggerthii DSM 20697 does not grow on alginate, but readily proliferates with a lag phase of a few hours in the presence of an endo-acting alginate lyase A1-I from the marine bacterium Sphingomonas sp. A1. The B. eggerthii lyases are both exo-acting and while BePL6 is strictly G-block specific, BePL17 prefers M-blocks. BeKdgF retained 10−27% activity in the presence of 0.1−1 mM EDTA. X-ray crystallography was used to investigate the three-dimensional structure of BeKdgF, based on which a catalytic mechanism was proposed to involve Asp102, acting as acid/base having pKa of 5.9 as determined by NMR pH titration. BePL6 and BePL17 cooperate in alginate degradation with BeKdgF linearizing producing 4,5-unsaturated monouronates. Their efficiency of alginate degradation was much enhanced by the addition of the A1-I alginate lyase. [ABSTRACT FROM AUTHOR]

In the pentose phosphate pathway, dehydroepiandrosterone (DHEA) uncompetitively inhibits glucose‐6‐phosphate dehydrogenase (G6PD), reducing NADPH production and increasing oxidative stress, which can influence the onset and/or progression of several diseases, including cancer. 2‐Deoxy‐D‐glucose (2‐DG), a glucose mimetic, competes with glucose for cellular uptake, inhibiting glycolysis and competing with glucose‐6‐phosphate (G‐6‐P) for G6PD activity. In this study, we report that DHEA‐α‐2‐DG (5), an α‐covalent conjugate of DHEA and 2‐DG, exhibits better anticancer activity than DHEA, 2‐DG, DHEA +2‐DG, and polydatin in MCF‐7 cells, and reduces NADPH/NADP+ ratio in cellular assays. In vitro enzyme kinetics and molecular docking studies showed that 5 uncompetitively inhibits human G6PD activity and binds to the structural NADP+ site but not to the catalytic NADP+ site. Further combining 5 with the FDA‐approved drug tamoxifen enhanced its cytotoxicity against MCF‐7 cells, suggesting that it could serve as a candidate for combination of drug strategies. [ABSTRACT FROM AUTHOR]

This study aims to investigate the potential impact of inhibitors targeting the papain-like protease (PLpro) of SARS-CoV-2 on viral replication and the host immune response. A mathematical model was developed to simulate the interaction among susceptible cells, infected cells, PLpro, and immune cells, incorporating data on PLpro inhibition. Through numerical simulations using MATLAB, the model parameters were estimated based on available statistical data. The results indicate that strategically positioned inhibitors could impede the virus’s access to host cellular machinery, thereby enhancing the immune response and gradually reducing susceptible and infected cells over time. The dynamics of the viral enzyme PLpro showed reduced activity with the introduction of the inhibitor, leading to a decline in viral replication. Moreover, the immune cell population exhibited functional recovery as the inhibitor suppressed PLpro activity. These findings suggest that inhibitors targeting PLpro may serve as therapeutic interventions against SARS-CoV-2 by inhibiting viral replication and bolstering the immune response. [ABSTRACT FROM AUTHOR]

Biological soil crusts (biocrusts) are crucial elements of desert ecosystems, exhibiting patchy distribution patterns across the soil surface and significantly impacting surface soil nutrient dynamics. However, the influence of biocrust patch units, serving as fundamental functional entities, on microbial nutrient restriction remains underexplored. This study conducted measurements on moss crust patches of varying sizes and subcrust soils. Stoichiometric analysis of extracellular enzyme activities (EEAs) related to carbon, nitrogen, and phosphorus was performed, along with vector and redundancy analyses to evaluate microbial nutrient limitation and key influencing factors. The findings reveal that both patch size and soil layer of biocrusts collectively influence soil nutrients and enzyme dynamics, with heightened enzyme activity observed in the crust layer. Vector analysis based on EEA stoichiometry indicates that moss crust patch size insignificantly impacts microbial nutrient restriction within the crust layer. However, microbial nitrogen restriction in the subcrust layer demonstrates a “single-peak” trend, initially increasing before gradually declining. This suggests that microorganisms in medium-sized crust patches exhibit peak activity, intensifying nutrient competition. This research underscores the pivotal role of biocrust patch units as fundamental functional entities, offering comprehensive insights into microbial metabolic constraints under crust cover. The findings underscore the significant implications of enzyme stoichiometric characteristics for desert land management and conservation. [ABSTRACT FROM AUTHOR]

In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (K m). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (k cat or V max). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of K m , k cat or V max without the need for authentic metabolite standards. This approach, using the carbon-14 [14C] labeled metabolite to calibrate the unlabeled metabolite (14C calibration method), combines radiometric with LC-MS/MS detection to generate both [14C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the 14C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using 14C calibration method and evaluated the enzyme kinetics of metabolite formation. The K m values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the 14C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis. [ABSTRACT FROM AUTHOR]

Ciliates are a well‐defined, species‐rich, versatile group of heterotrophic and mixotrophic protists. Their response to increased temperature during global warming is critical for the structure and functioning of freshwater food webs.I conducted a meta‐analysis of the literature from field studies and experimental evidence to assess the parameters characterising the thermal response of freshwater ciliates. The shape of the thermal performance curve predicts the ciliates' survival at supraoptimal temperatures (i.e., the width of the thermal safety margin).The ciliates' typical thermal safety margin is c. 5°C. More than two‐thirds of the freshwater ciliates dwelling permanently or occasionally in the pelagial cannot survive at temperatures exceeding 30°C. Likewise, cold‐stenothermic species, which represent a significant fraction of euplanktonic ciliates, cannot survive by evolutionary adaptation to rapidly warming environments.The statistical analysis revealed that the ciliates' thermal performance is affected by their planktonic lifestyle (euplanktonic vs. tychoplanktonic), ability to form cysts and nutritional ecology. Bactivorous ciliates have the widest temperature niche, and algivorous ciliates have the narrowest temperature niche.Phenotypic plasticity and genetic variation, favouring the selection of pre‐adapted species in a new environment, are widespread among freshwater ciliates. However, the lack of evidence for the temperature optima and imprecisely defined tolerance limits of most species hampers the present analysis. The extent of acclimation and adaptation requires further research with more ciliate species than the few chosen thus far.Recent eco‐evolutionary experimental work and modelling approaches have demonstrated that the ciliates' thermal responses follow general trends predicted by the metabolic theory of ecology and mechanistic functions inherent in enzyme kinetics. The present analysis identified current knowledge gaps and avenues for future research that may serve as a model study for other biota. Thermal adaptation may conflict with adaptation to other stressors (predators, food availability, pH), making general predictions on the future role of freshwater ciliates in a warmer environment difficult, if not impossible, at present. [ABSTRACT FROM AUTHOR]

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost‐effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis–Menten and Lineweaver–Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH‐dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost‐effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources. [ABSTRACT FROM AUTHOR]

Serine proteases have been receiving special attention from the industrial point of view, due to their thermo-stable properties and activity over wide ranges of pH. In the present investigation, a serine protease (49.3 kDa) from jackfruit latex has been purified using chromatographic techniques. Fold purification of the serine protease in the final purification step was 92.41 with 24% yield. The protease is completely inhibited by PMSF, a serine protease-specific inhibitor at a minimal concentration (100 µM). The purified serine protease was immobilized with sodium alginate (2.5%) and calcium chloride (0.3 M) solution. The kinetic studies of immobilized enzyme showed stable activity up to pH 7, and can withstand temperatures up to 45 °C. The immobilization process improves the catalytic efficiency of the enzyme over purified soluble enzyme (for Kcat 1.4 times and for Kcat/Km 1.21 times). Our results depicted that alginate mediated immobilization of serine protease greatly improves the pH and temperature optima which broadens the scope of usage of the enzyme further. [ABSTRACT FROM AUTHOR]

Peroxyacid synthesis is the first step in Prilezhaev epoxidation, which is an industrial method to form epoxides. Motivated by the development of a kinetic model as a tool for solvent selection, the effect of solvent type and acid chain length on the lipase‐catalyzed peroxyacid synthesis was studied. A thermodynamic activity‐based ping‐pong kinetic expression was successfully applied to predict the effect of the reagent loadings in hexane. The activity‐based reaction quotients provided a prediction of solvent‐independent equilibrium constants. However, this strategy did not achieve satisfactory estimations of initial rates in solvents of higher polarity. The lack of compliance with some assumptions of this methodology could be confirmed through molecular dynamics calculations i.e. independent solvation energies and lack of solvent interaction with the active site. A novel approach is proposed combining the activity‐based kinetic expression and the free binding energy of the solvent with the active site to predict kinetics upon solvent change. Di‐isopropyl ether generated a strong interaction with the enzyme's active site, which was detrimental to kinetics. On the other hand, toluene or limonene gave moderate interaction with the active site rendering improved catalytic yield compared with less polar solvents, a finding sharpened when peroctanoic acid was produced. [ABSTRACT FROM AUTHOR]

BACKGROUND: The industrial‐scale application of enzymes faces obstacles due to elevated costs and difficulties in stability and reuse. In this study, magnetic spent coffee grounds, an ecotoxic waste, have been utilized successfully for the first time to immobilize β‐glucosidase to overcome these challenges. RESULTS: The spent coffee grounds were magnetized and amine‐functionalized, followed by characterization using various techniques. Under optimized conditions, forming an imine bond between the functionalized support and β‐glucosidase resulted in a 62% immobilization yield (92.81 mg g−1 enzyme loading) and 12.5 U mg−1 activity after immobilization. A relatively small kinetic change was observed in the Km value (902 to 946 μmol L−1) after immobilization, suggesting minimal hindrance by AMSCG3 on substrate access or product release. Moreover, Glu@AMSCG3 showed exceptional stability (>90% residual activity) within a pH range of 3 to 6 after 2 h of incubation at 25 °C. A residual activity of 87.94% was maintained even at 80 °C and pH 5 after 2 h of incubation compared to the free β‐glucosidase, which showed only 6.5% residual activity at the same temperature. When cellobiose was hydrolyzed using Glu@AMSCG3 under optimum conditions, 91.33% cellobiose conversion was achieved initially, and over 79% conversion was maintained for 10 reusability cycles. CONCLUSION: The improved stability of β‐glucosidase after covalent immobilization on amine‐modified magnetically separable spent coffee grounds indicates their potential as a support matrix for application in enzyme immobilization. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

The intracellular environment displays complex dynamics influenced by factors such as molecular crowding and the low Reynolds number of the cytoplasm. Enzymes exhibiting active matter properties further heighten this complexity which can lead to memory effects. Molecular simulations often neglect these factors, treating the environment as a "thermal bath" using the Langevin equation (LE) with white noise. One way to consider these factors is by using colored noise instead within the generalized Langevin equation (GLE) framework, which allows for the incorporation of memory effects that have been observed in experimental data. We investigated the structural and dynamic differences in Shikimate kinase (SK) using LE and GLE simulations. Our results suggest that GLE simulations, which reveal significant changes, could be utilized for assessing conformational motions' impact on catalytic reactions. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) has become a growing concern to global public health, being at the forefront of acute disorders and causes of mortality across the globe. Clinically approved drugs that are currently being used are faced with severe side effects, consequently necessitating the development of new drugs with no/fewer side effects and improved pharmacological potency. Herein, we report a rapid and efficient synthesis of thiazolidinone Schiff bases (2a-2t) from benzylidenehydrazines and thioglycolic acid under neat conditions through ultra-sonication. All the synthesized compounds were obtained in exceptional yields (89–95%) and confirmed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as High-resolution mass spectrometry (HRMS). The synthesized compounds were then evaluated for their antidiabetic activity through α-glucosidase and α-amylase inhibitory potentials and their antioxidant activity through Nitric Oxide (NO), 2,2′-diphenyl-1-picrylhydrazyl (DPPH), and Ferric reducing antioxidant power (FRAP) assays. Among them, 2q (IC50 = 96.63 μM) and 2h (IC50 = 125.27 μM) emerged as the most potent derivatives against α-amylase relative to reference drug acarbose (IC50 = 131.63 µM), respectively. Antioxidant evaluation further revealed that the synthesized derivatives were excellent NO scavengers disclosing 2n (IC50 = 44.95 µM) as the most potent derivative. Moreover, in silico ADME calculations predicted these compounds to have excellent drug-like properties. Kinetic studies disclosed the mode of α-amylase inhibition as competitive while molecular docking studies of the most active derivatives performed into the binding active site of human pancreatic α-amylase enzyme deciphered their ligand-protein interactions that explicated their observed experimental potencies. [ABSTRACT FROM AUTHOR]

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s-1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD. KEY POINTS: • Anaerobic fungi host a wealth of industrially useful enzymes but are understudied. • P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes. • CelDs kinetics do not change with domain fusion, exhibiting high modularity.

Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders. Current pharmacological interventions targeting CTSL have demonstrated potential in reducing body weight gain, serum insulin levels, and improving glucose tolerance. However, the clinical application of CTSL inhibitors remains limited. In this study, we used a combination of artificial intelligence and experimental methods to identify new CTSL inhibitors from natural products. Through a robust deep learning model and molecular docking, we screened 150 molecules from natural products for experimental validation. At a concentration of 100 µM, we found that 36 of them exhibited more than 50 % inhibition of CTSL. Notably, 13 molecules displayed over 90 % inhibition and exhibiting concentration-dependent effects. The molecular dynamics simulation on the two most potent inhibitors, Plumbagin and Beta-Lapachone, demonstrated stable interaction at the CTSL active site. Enzyme kinetics studies have shown that these inhibitors exert an uncompetitive inhibitory effect on CTSL. In conclusion, our research identifies Plumbagin and Beta-Lapachone as potential CTSL inhibitors, offering promising candidates for the treatment of metabolic disorders and illustrating the effectiveness of artificial intelligence in drug discovery.

Enzymes can produce high-quality food with low pollution, high function, high acceptability, and medical aid. However, most enzymes, in their native form, do not meet the industrial requirements. Sequence-based and structure-based methods are the two main strategies used for enzyme modification. Molecular Dynamics (MD) simulation is a sufficiently comprehensive technology, from a molecular perspective, which has been widely used for structure information analysis and enzyme modification. In this review, we summarize the progress and development of MD simulation, particularly for software, force fields, and a standard procedure. Subsequently, we review the application of MD simulation in various food enzymes for thermostability and catalytic improvement was reviewed in depth. Finally, the limitations and prospects of MD simulation in food enzyme modification research are discussed. This review highlights the significance of MD simulation and its prospects in food enzyme modification. [ABSTRACT FROM AUTHOR]

Malaria remains a major health issue worldwide that affects many people, particularly in developing nations. Since, the malarial parasite has developed resistance against nearly every antimalarial drug now in use, it is imperative to search for novel antimalarial medications. Toxins produced by skin glands of toads have been shown to possess antiparasitic properties against a variety of protozoan parasites because of the bufadienolides they contain. Even though several studies have been conducted to show that toad skin secretions have antimalarial properties, very little information is known about the precise mechanism by which they work against Plasmodium infection. Thus, the goal of this study was to evaluate the antiplasmodial activity of crude skin extracts from Common Asian Toads, Duttaphrynus melanostictus, of different sizes and illustrate how they work against Plasmodium falciparum 3D7 cells. The findings demonstrated a negative correlation between the toad size and percent yield of the extracts. HPTLC and UPLC-MS/MS analysis of the extracts exhibited varied composition of bufadienolides depending on the size of the animal. The extract obtained from small toads containing resibufagin and marinobufagin lactate demonstrated highest antiplasmodial activity and showed lowest cytotoxicity on peripheral blood mononuclear cells. It was discovered that the extract was effective against the trophozoite stage of the parasite. The extract was reported to inhibit Na+/H+ ATPase of Plasmodium by binding to sodium-enzyme complex at ATP binding site. The study offers baseline data that can be used to assess the antimalarial potential of individual components in the skin extract derived from small toads. [ABSTRACT FROM AUTHOR]

Abstract Background Aldehyde dehydrogenase 2 (ALDH2) is critical for alcohol metabolism by converting acetaldehyde to acetic acid. In East Asian descendants, an inactive genetic variant in ALDH2, rs671, triggers an alcohol flushing response due to acetaldehyde accumulation. As alcohol flushing is not exclusive to those of East Asian descent, we questioned whether additional ALDH2 genetic variants can drive facial flushing and inefficient acetaldehyde metabolism using human testing and biochemical assays. Methods After IRB approval, human subjects were given an alcohol challenge (0.25 g/kg) while quantifying acetaldehyde levels and the physiological response (heart rate and skin temperature) to alcohol. Further, by employing biochemical techniques including human purified ALDH2 proteins and transiently transfected NIH 3T3 cells, we characterized two newly identified ALDH2 variants for ALDH2 enzymatic activity, ALDH2 dimer/tetramer formation, and reactive oxygen species production after alcohol treatment. Results Humans heterozygous for rs747096195 (R101G) or rs190764869 (R114W) had facial flushing and a 2-fold increase in acetaldehyde levels, while rs671 (E504K) had facial flushing and a 6-fold increase in acetaldehyde levels relative to wild type ALDH2 carriers. In vitro studies with recombinant R101G and R114W ALDH2 enzyme showed a reduced efficiency in acetaldehyde metabolism that is unique when compared to E504K or wild-type ALDH2. The effect is caused by a lack of functional dimer/tetramer formation for R101G and decreased Vmax for both R101G and R114W. Transiently transfected NIH-3T3 cells with R101G and R114W also had a reduced enzymatic activity by ~ 50% relative to transfected wild-type ALDH2 and when subjected to alcohol, the R101G and R114W variants had a 2-3-fold increase in reactive oxygen species formation with respect to wild type ALDH2. Conclusions We identified two additional ALDH2 variants in humans causing facial flushing and acetaldehyde accumulation after alcohol consumption. As alcohol use is associated with a several-fold higher risk for esophageal cancer for the E504K variant, the methodology developed here to characterize ALDH2 genetic variant response to alcohol can lead the way precision medicine strategies to further understand the interplay of alcohol consumption, ALDH2 genetics, and cancer.

The dry aerial part of Panax japonicus var. major is called “Zhuzishen Ye”, it is a characteristic Chinese herbal medicine in Qinba area. In order to rationally develop and utilize “Zhuzishen Ye” and clarify its chemical constituents, the main chemical constituents of the saponin fractions from “Zhuzishen Ye” were analyzed with HPLC, the inhibitory activities and inhibition types of the saponin fractions of “Zhuzishen Ye” on lipase were determined, and the lipase inhibition mechanisms and hypolipidemic effects were verified with molecular docking and animal experiments. The results were as follows: (1) The chemical constituents of saponin fractions from “Zhuzishen Ye” were 20(S)-ginsenoside Rg2, 20(R)-ginsenoside Rg2, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd and ginsenoside Rh2. (2) The saponin fractions of “Zhuzishen Ye” and 20(R)-ginsenoside Rg2 had strong inhibitory effects on lipase with IC50 values of 0.14 and 2.30 μmol·L-1, respectively. (3) The saponin fractions of “Zhuzishen Ye”, 20(R)-ginsenoside Rg2 and ginsenoside Rb3 were all reversible inhibition, and the inhibition type was non-competitive inhibition. (4) The binding of ligands to ARG337B, ASP331B and ILE248B residues might help to improve the lipase inhibitory activity of ligands. (5) Saponins of “Zhuzishen Ye” could significantly reduce the content of cholesterol and triglyceride in serum of hyperlipidemia mice. This study provides the theoretical references for the further development and utilization of “Zhuzishen Ye”.

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. Key points: • A novel neuraminic acid synthase was characterized. • The effect of cofactors on NeuS activity was elucidated. • Kinetic and stability characterization of two neuraminic acid synthases was performed. [ABSTRACT FROM AUTHOR]