Caffeic acid O-methyltransferase (COMT) is an essential enzyme that catalyzes the biosynthesis of lignin monomer units. Foxtail millet (Setaria italica) possesses three COMT-encoding genes. However, only SiCOMT1 and SiCOMT2 are considered expressed. This study investigates the characteristics of the two COMT-encoding genes across nine Indonesian foxtail millet genotypes. Phylogenetic analysis revealed that SiCOMT1 is closely related to N-methyltransferase genes, which are not involved in the lignin biosynthesis pathway. Meanwhile, SiCOMT2 is closely related to O-methyltransferase genes involved in lignin biosynthesis. SiCOMT2 from nine Indonesian foxtail millet genotypes exhibits 15 synonymous and three non-synonymous SNPs. SiCOMT2 amino acid showed Ala67Thr and Pro72Ala variations within the methyltransferase dimerization domain, and Glu146Asp within the O-methyltransferase domain. Among these, the Pro72Ala substitution is predicted to reduce the structural stability of the encoded protein. These findings suggest that SiCOMT2 may serve as a promising target for future genetic research and crop improvement strategies aimed at enhancing biomass quality by modifying lignin content and composition.

DNA Topoisomerase II Mutations in Cancer: Structural Impact and Drug Response in High-grade Serous Ovarian Carcinoma.

Proteases are essential enzymes that, through the breakdown of proteins, regulate many aspects of tissue homeostasis including barrier function, cellular signaling, and tissue repair mechanisms in organisms. Disease gene discovery in a number of monogenic skin diseases has deepened the knowledge of how proteases and protease inhibitors can regulate skin homeostasis, keratinocyte desmosome-mediated cell adhesion, and epidermal barrier function. This short review details the association of protease dysregulation with monogenic skin diseases, postulated disease mechanisms, and emerging therapeutic strategies.

Flavin reductases in two-component systems: Mechanistic insights, structural classification, and biotechnological advances.

Probing the conserved catalytic mechanism of ThiL protein in pathogenic Leptospira species: An in silico strategy for inhibitor discovery to combat leptospirosis.

Structure and mechanistic basis of NrdR, a bacterial master regulator of ribonucleotide reduction.

Proteomic profiling reveals the mechanism of ginsenoside synthesis in Panax notoginseng.

ABSTRACTThe increasing prevalence of persistent and difficult‐to‐treat infections has intensified interest in natural compounds able to act on both planktonic bacteria and biofilm structures. Tithonia diversifolia, a species rich in bioactive metabolites such as tagitinin C, has emerged as a promising source of antimicrobial agents. This study evaluated the antibacterial and antibiofilm activities of a tagitinin C‐rich leaf extract and potential mechanisms of action. The extract was obtained by supercritical CO2 extraction, chemically characterized by HPLC, and assessed against Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa using disk diffusion, broth microdilution, and molecular docking assays. Antibiofilm activity was investigated against a biofilm‐forming S. aureus strain using coverslip disruption assays, optical microscopy, and scanning electron microscopy. The extract showed strong antimicrobial activity against S. aureus (MIC/MBC: 0.039 mg/mL), intermediate inhibition of E. faecalis (1.25 mg/mL), and moderate effects on P. aeruginosa (5 mg/mL). It also promoted significant biofilm disruption (66.2% ± 4.1%), with microscopy revealing reduced bacterial density and altered structural organization. Docking results demonstrated favorable binding affinities with MurA, MurE, and MurG, suggesting stable interactions with essential enzymes involved in peptidoglycan synthesis. Overall, the findings highlight the therapeutic potential of T. diversifolia extract against resistant microorganisms and biofilm‐associated infections.

Design, synthesis and biological evaluation of novel quinolinone derivatives as DprE1 inhibitors against Mycobacterium tuberculosis.

The use of antibiotics as growth promoters in livestock production has contributed to the emergence and spread of antimicrobial resistance (AMR), posing a significant global public health threat specifically from the projected mortality burden. Although many countries have restricted the non-therapeutic use of antibiotics, practical and effective alternatives are still required to maintain livestock productivity. This scoping review examines the current evidence on non-antibiotic compounds evaluated as growth-promoting agents in livestock production. The primary objective of this search was to generate a comprehensive list of commonly applied alternatives to antibiotics used as growth promoters in livestock systems. A search was conducted in the CAB Abstracts, Web of Science Core Collection, and AGRICOLA databases. Prior to the scoping review, an initial list of alternatives to antibiotic components was generated through a screening of selected scientific sources and subsequently verified using Google Scholar for the period 2010–2025. This list included brief descriptions of each component, which were used to inform the keyword strategy for the scoping review. Eligible studies were screened in accordance with PRISMA-ScR guidelines, and data were extracted on compound type, livestock species, geographic region, and reported performance outcomes. The alternatives identified included probiotics and prebiotics, phytogenic compounds and essential oils, enzymes and organic acids, vaccines and immunostimulants, bacteriophages, and competitive exclusion products. A total of 1230 records were retrieved and imported into Zotero for reference management. After removal of duplicate records using Zotero’s built-in deduplication tool, 377 unique records remained for screening. Overall, these compounds demonstrated variable effects on feed efficiency, weight gain, and gut health. However, most studies were limited in scale, duration, and methodological consistency. As a result, comprehensive comparative trials and large-scale field evaluations are needed to support evidence-based policy recommendations and the sustainable implementation of alternatives to antibiotics in livestock production systems. Our findings identified six major categories that represent the most frequently reported alternatives to antibiotic growth promoters. Although probiotics, phytogenic, and organic acids were the most extensively studied, substantial heterogeneity in trial design, dosage, and production systems limited meaningful cross-comparisons. In addition, most studies focused on poultry and swine, with comparatively fewer investigations involving ruminant species. This scoping review was not intended to evaluate the efficacy or practical applicability of these alternatives; such assessments require further standardized and extensive studies before recommendations for their widespread application can be made.

ABSTRACT The global spread of multidrug‐resistant tuberculosis (MDR‐TB) necessitates the discovery of novel chemotypes with strong inhibitory activity against essential bacterial enzymes. Decaprenylphosphoryl‐β‐D‐ribose 2′‐epimerase (DprE1), indispensable for cell wall biosynthesis in Mycobacterium tuberculosis , represents a validated but underexploited target. In this work, we employed an integrated computational pipeline combining pharmacophore modeling, validated docking, large‐scale virtual screening of ∼538,000 compounds, molecular dynamics simulations, MM‐PBSA free energy calculations, and machine learning–based antimicrobial prediction. This approach identified 18 previously unreported scaffolds, three of which demonstrated highly stable interactions with key catalytic residues (CYS387, LYS418, ILE131) during 200 ns molecular dynamics simulations. Binding free energy analysis confirmed favorable energetics, with Hit 1 (−28.5 kcal/mol) and Hit 3 (−24.3 kcal/mol) outperforming the reference inhibitor G1T. Machine learning predictions further suggested sub‐nanomolar MIC values for these leads, highlighting their promising anti‐tubercular potential. Scaffold novelty analysis indicated that the top hits are chemically distinct from known inhibitors. Collectively, this study reports novel and biologically relevant DprE1 inhibitors that merit further optimization and experimental validation as next‐generation anti‐TB therapeutics.

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) are widely used to modulate gene expression in basic research and therapy. Within cells, these ASOs seed nuclear structures with unclear functions and consequences. At DNA breaks, endogenous nucleotide polymers drive the assembly of biomolecular condensates that recruit repair proteins, but the underlying mechanism(s) and effects on repair enzyme activation are poorly understood. Here, we show that ASOs bind to DNA-PKcs, ATM, and PARP1, triggering phase separation and formation of nuclear condensates containing ASOs and these essential repair enzymes. Condensates assembly is stimulated by ASO concentration and ATM activity, while limited by DNA-PKcs activity. Notably, these condensates become enzymatically active and erroneously elicit the DNA damage response in the absence of DNA damage, activating cell cycle checkpoints, disturbing endogenous repair and causing accumulation of toxic DNA lesions. These findings uncover mechanisms for ASO toxicity and the activation of DNA repair enzymes by nucleotide polymers.

DNA topoisomerase-IA is an essential enzyme that relaxes supercoiled DNA by introducing transient single-strand breaks through a covalent phosphorylated tyrosine (PTR) intermediate. This cleavage occurs when the active-site tyrosine of dTopo-IA forms a covalent bond with the DNA phosphate backbone, resulting in PTR formation. Although dTopo-IA is believed to mediate strand passage via an enzyme-induced DNA gate, the actual opening of this gate has not been demonstrated experimentally or theoretically. To address this gap, we employed 200-nanosecond (ns) molecular dynamics (MD) simulations using AMBER18 to explore the catalytic mechanism and conformational dynamics of dTopo-IA. Important parameters like RMSD, RMSF, the number of hydrogen bonds, hydrogen bond distances, the radius of gyration (RoG), binding free energy, solvent-accessible surface area (SASA), and per-residue pair-wise decomposition energy were analyzed. Our simulations revealed that the bond between PTR and nucleotide acts as a gatekeeper, regulating the opening and closing of the DNA gate critical for strand passage. MD trajectories clearly demonstrate that gate opening and strand passage occur only after the formation of the covalent bond between PTR and the C5′ atom of the DNA strand. Additionally, we investigated how topoisomerase selectively binds single-stranded DNA in the presence of double-stranded DNA to initiate its catalytic function. The enzymatic roles of residues Gln-223, Arg-533, and Lys-117 were also elucidated in the process. This provides a novel and deeper understanding of the enzyme’s mechanism, which has been challenging to capture through experimental techniques alone, and potentially aids the development of targeted anticancer therapies by disrupting DNA replication in cancer cells.

The SARS-CoV-2 papain-like protease (PLpro) is an essential viral enzyme that promotes viral polyprotein processing while simultaneously suppressing the host innate immune response, which makes it a primary target for developing antiviral drugs. The present study employs a comprehensive approach integrating untargeted metabolomic profiling, in silico molecular docking and dynamics simulations, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) energetic assessments, and biochemical enzyme assays. This integrated method aims to discover natural PLpro inhibitors from two ethnomedicinal plants, Lippia javanica and Acorus calamus, which have long been utilized in African traditional medicine to treat respiratory diseases. Comprehensive metabolite profiling using untargeted Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and Global Natural Products Social (GNPS) molecular networking revealed flavonoid glucuronides and phenylpropanoid derivatives as the major constituents in both plant species. In situ histochemical staining further offered spatial validation of phenolic- and lignin-associated tissues, supporting the phenolic-dominated molecular families detected by GNPS molecular networking. In silico evaluation of six selected compounds demonstrated spontaneous and thermodynamically favorable binding to PLpro, with ΔG_bind values ranging from -5.63 to -6.43 kcal/mol. Catechin-7-glucoside emerged as the lead compound, establishing multiple hydrogen bond networks with Asp164, Gln269, Tyr264, and Asn267, supplemented by hydrophobic engagement with Pro247 and Pro248, and π-π stacking with the blocking loop 2 (BL2 loop). Molecular dynamics simulations confirmed the stability of the protein-ligand complexes. Biochemical enzyme assays confirmed concentration-dependent inhibition of PLpro proteolytic and deubiquitinating activity by both crude plant extracts and isolated bioactive compounds. However, S-adenosyl-methionine showed comparatively high PLpro proteolytic activity (IC50 5.872 µM) compared to catechin-7-glucoside, with an IC50 of 7.493 µM, exhibiting efficacy similar to the reference inhibitor GRL0617. Both the extracts of L. javanica and A. calamus have shown significant inhibitory activity while maintaining cell viability in Human embryonic kidney 293T cell (HEK293T) culture models, indicating a favorable safety profile of the tested concentrations. Based on these results, catechin-based polyphenols and phenylpropanoid derivatives appear as promising lead compounds for the development of PLpro inhibitors. To progress toward therapeutic use, further work is necessary in pharmacokinetics, structural optimization, and antiviral validation in cell models.

Oral squamous cell carcinoma (OSCC) is a highly aggressive malignancy characterized by frequent recurrence and metastasis, which poses a significant global health problem. One of the prominent hallmarks of cancer is glucose metabolic reprogramming, wherein glycolysis is preferred over oxidative phosphorylation for macromolecule biosynthesis and energy production, even in the presence of oxygen. Non-coding RNAs (ncRNAs) are defined as a class of RNAs that are not translated into proteins, which include microRNAs, long non-coding RNAs, and circular RNAs. Recent studies have found that ncRNAs are crucial in regulating glycolysis in OSCC, wherein they reshape the metabolic landscape by modulating the expression of glucose transporters, essential enzymes, and transcription factors, ultimately influencing tumorigenesis. This comprehensive review systematically summarizes the regulatory mechanisms of ncRNAs involved in glucose metabolic reprogramming in OSCC, evaluates their potential as diagnostic biomarkers and therapeutic targets, and identifies clinically relevant ncRNAs through an integrative analysis of patient-derived data. These insights provide a mechanistic understanding of the metabolic alterations that drive progression in OSCC, as well as knowledge that can facilitate the development of clinically translatable targeted interventions for this aggressive malignancy.

A fundamental driver of biological diversification is the evolution of reproductive barriers between species. Instability and mis-regulation of repetitive DNA underlie numerous post-zygotic reproductive barriers, yet the molecular mechanisms are unknown. A long-studied genetic incompatibility between Drosophila melanogaster and D. simulans arises from mis-segregation of the D. melanogaster-specific 359bp DNA satellite in hybrid embryos. Here we report that the D. simulans version of the essential enzyme Topoisomerase II/Top2 causes this lethal incompatibility. Combining interspecies gene swaps with cell biology and genetics revealed that D. simulans-specific adaptive divergence of Top2 DNA-interacting domains prevents the resolution of 359bp-induced topological stress. Our findings demonstrate that species-specific DNA satellite topology requires species-specific molecular machinery and that even vital housekeeping genes can underlie reproductive isolation between closely related species.

Emerging mosquito-borne flaviviruses, such as Dengue virus (DENV) and Zika virus (ZIKV), pose major global public health threats due to their geographic expansion, climate change, and the absence of effective antiviral therapies. Antiviral development against these pathogens has primarily focused on two complementary strategies. On the one hand, the blocking of viral replication by directly inhibiting essential viral enzymes, and on the other, enhancing the host's innate immune defenses via targeted activation of intracellular antiviral pathways. Among the viral proteins required for replication, the NS2B-NS3 protease complex is one of the most conserved and druggable targets, prompting extensive efforts to design both covalent and non-covalent inhibitors. Covalent inhibitors, such as boronic acids, aldehydes, trifluoromethyl ketones, phenoxymethylphenyl derivatives, and α-ketoamides, form irreversible or slowly reversible bonds with the catalytic serine residue (Ser 135), producing long-lasting and high-affinity suppression of protease activity. In parallel, several classes of non-covalent, particularly allosteric, inhibitors have emerged as promising alternatives with improved specificity and reduced off-target reactivity. A complementary antiviral strategy involves the use of agonists of key innate immune sensors such as TLRs, RIG-I, and the cGAS-STING axis, which mediate the release of interferons (IFNs). This review brings together current knowledge on these two mechanistically distinct yet convergent approaches, highlighting how both can ultimately restrict flavivirus replication. Future opportunities involving modified peptide scaffolds, advanced delivery systems, and drug-repurposing strategies are finally discussed for the development of next-generation therapeutics against DENV and ZIKV.

Fungal inositol phosphorylceramide (IPC) synthase is an essential enzyme complex that catalyzes a critical step in sphingolipid biosynthesis. It is the molecular target of potent antifungal aureobasidin A (AbA). Despite its therapeutic relevance, the lack of structural and mechanistic insights into IPC synthase function and inhibition has impeded rational antifungal drug development. Here, we present cryo-EM structures of Saccharomyces cerevisiae IPC synthase in two distinct functional states: a ceramide-bound form and an AbA-inhibited complex. Our study reveals a conserved heterodimeric architecture formed by Aur1 and Kei1, stabilized through extensive protein-protein and lipid-mediated interactions. Within catalytic Aur1, we identify a membrane-embedded reaction chamber harboring a conserved H-H-D catalytic triad (H255, H294, and D298) essential for IPC synthesis. Structural comparisons illuminate the mechanism of ceramide recognition and reveal how AbA acts as a competitive inhibitor by occupying the substrate-binding pocket. Further analyses identify key residues involved in AbA binding and explain the molecular basis of drug resistance. Together, these findings advance the mechanistic understanding of fungal IPC biosynthesis and inhibition, and establish a foundation for developing new antifungal drugs targeting IPC synthase.

β-Glucosidases are essential enzymes in plant cell wall metabolism and have diverse biotechnological applications, including cellulose degradation and prebiotic oligosaccharide synthesis. Td2F2, a glycoside hydrolase family 1 (GH1) β-glucosidase derived from a compost metagenome, exhibits a unique preference for sophorose. However, the molecular basis of this specificity remains unclear. In this study, we determined high-resolution crystal structures of Td2F2 in complex with sophorose (1.64 Å) and laminaribiose (1.16 Å) using sodium malonate as a cryoprotectant. Structural analysis, complemented by molecular dynamics simulations, revealed a distinct subsite +1′, where Asn223, Thr225, Glu296, and Arg325 form hydrogen bonds with the reducing-end glucose of sophorose, stabilizing an alternative, nonproductive binding mode adjacent to the catalytic subsites. Site-directed mutagenesis confirmed that residues in subsite +1′ are critical for substrate specificity. Guided by structural insights, we designed T225N and E296D mutants, which exhibited enhanced hydrolytic activity toward sophorose. To further investigate the transglycosylation potential of Td2F2, we characterized its dynamic product profile, ranging from disaccharides to tetrasaccharides, using porous graphitic carbon liquid chromatography–orbitrap tandem mass spectrometry. When p-nitrophenyl β-D-glucopyranoside and glucose were used as substrates, Td2F2 preferentially formed β-1→2 and β-1→3 linkages. These findings provide structural evidence that the subsite +1′ is a “waiting position” in the GH1 β-glucosidase, offering novel insights into its role in hydrolysis and transglycosylation selectivity. This structural and functional framework paves the way for future GH1 enzyme engineering and expanded biotechnological applications.

Among the plethora of RNA modifications, 5-methylcytosine (m5C) has held substantial roles in some biological processes, yet its impact on inflammation remains largely uncharted. Here, we reported an m5C-related epitranscriptomic regulatory axis in inflammatory pathogenesis. Bacterial lipopolysaccharides (LPS) challenge induces marked elevation in RNA m5C abundance as well as the expression of NOP2/Sun RNA methyltransferase 2 (NSUN2), an essential enzyme catalyzing m5C modification. Nsun2 deficient mice showed significantly reduced inflammatory response in the models of systemic inflammation and local pulpitis compared with wild-type control mice. Mechanistically, NSUN2 installs m5C modifications on interleukin 1 beta (IL1B) mRNA. These modifications are selectively recognized by the m5C reader Y-box binding protein 1 (YBX1), thereby enhancing transcript stability and promoting substantial elevation of IL1B, which might further drive inflammation cascades. Our research highlights a pivotal role of NSUN2-mediated m5C RNA modification in regulating inflammatory responses, offering fresh perspectives for the treatment of inflammatory diseases.