Search Results (593)

To address the issues of low degradation efficiency in anaerobic bacteria targeting microcystin-LR (MC-LR) and the limitations of traditional genetic modification methods, this study applied ARTP mutagenesis technology for the first time to genetically engineer anaerobic MC-LR-degrading bacteria using the Citrobacter sp. D03 isolated from Lake Taihu sediments as the parental strain. Through three rounds of ARTP-induced mutagenesis and directed screening, a highly efficient mutant, C29, was obtained. The results indicate that the optimal mutagenesis conditions are 60 s, with a lethality rate of 90.15%; the anaerobic degradation efficiency of MC-LR by mutant strain C29 was 14% higher than that of the wild-type strain, and the genetic efficiency remained stable after consecutive passages. Whole-genome resequencing of the new strain C29 revealed 10 mutation sites, involving key genes such as toxD, cheA, murI, and mobB. qRT-PCR validation showed that the expression of genes related to sulfur metabolism, cell wall synthesis, molybdenum cofactor synthesis, and environmental adaptation was significantly upregulated. The study confirms that ARTP mutagenesis can effectively enhance the degradation capacity of anaerobic degrading bacteria toward MC-LR through polygenic synergistic regulation, providing both microbial resources and a theoretical basis for in situ anaerobic bioremediation of MC-LR pollution in eutrophic water bodies. Full article

Background/Objectives: Symmetrical dual-site acetylcholinesterase (AChE) inhibitors offer a compelling strategy to mitigate mutational drug resistance, yet static modeling fails to capture induced-fit dynamics under mutational stress. Methods: Here, a 100,000-compound virtual library was filtered using a machine learning-based QSAR classification pipeline. A strict, empirically calibrated Jaccard applicability domain filter (AD = 0.823) eliminated topological anomalies, yielding a robust cross-validation accuracy (ROC-AUC: 0.80 ± 0.05; independent test MCC: 0.61). Multi-parameter ADMET and shape screening prioritized unique chemotypes to probe the 20 Å enzyme gorge. All-atom explicit-solvent molecular dynamics simulations were coupled with 150 ns enhanced-sampling Metadynamics along two orthogonal collective variables (gorge depth and ligand orientation) to map out the free energy surfaces under mutational stress. Results: Symmetrical probes suffered catastrophic unbinding upon anchor loss. Conversely, the symmetrical core of Lead Compound 1631 demonstrated extraordinary structural resilience. In silico site-directed mutagenesis (W86A and W286A) triggered a thermodynamic locking effect; the W86A mutant forced the complex into a deeper energetic well (ΔG_min = 9.23 ± 1.98 kJ/mol) than the wild-type state (5.26 ± 1.69 kJ/mol). MM/GBSA decomposition confirmed an active electrostatic-solvation compensation mechanism along a “solvation see-saw” diagonal (ΔΔG_total = +1.59 kcal/mol). Finally, Dynamic Cross-Correlation Matrix analysis quantified a mechanical inversion of the CAS-PAS axis into an anti-correlated clamping mode (−0.04) that locked the ligand bridge in place. Conclusions: These results demonstrate that symmetrical dual-site targeting, combined with dynamic thermodynamic locking, provides a resilient framework to overcome mutational resistance in AChE inhibitors. Full article

N-acetylornithine aminotransferase (AcOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a key transamination step in arginine biosynthesis. In cyanobacteria, arginine metabolism is closely associated with nitrogen assimilation and storage, yet biochemical information on cyanobacterial AcOATs remains limited. In this study, the AcOAT encoded by the cce_3094 gene from Crocosphaera subtropica ATCC 51142 (CsAcOAT) was cloned, heterologously expressed, purified, and systematically characterized. Recombinant CsAcOAT was obtained as a soluble protein with an apparent molecular mass of approximately 43 kDa. Steady-state kinetic analysis showed that CsAcOAT catalyzed transamination between N-acetylornithine (AcOrn) and α-ketoglutarate (α-KG), with apparent K_M values of 0.17 ± 0.03 mM for AcOrn and 0.020 ± 0.003 mM for α-KG, indicating a higher affinity for α-KG. The enzyme exhibited optimal activity at pH 8.5 and 30 °C, retained relatively high activity over a broad temperature range of 0–50 °C, and was activated by Zn²⁺ and Co²⁺ but inhibited by Ni²⁺. Structural analysis based on homology modeling, molecular docking, and molecular dynamics simulations suggested a conserved PLP-dependent aminotransferase fold and a stable binding mode for the PLP-AcOrn complex in the active-site pocket. Site-directed mutagenesis further demonstrated that Gly114, Asp239, Lys268, and Thr296 are indispensable for catalytic activity, whereas Ser113, Ala115, and Gln242 make important contributions to catalytic turnover and cofactor-assisted catalysis. These results provide biochemical and structural characterization of CsAcOAT, expand current knowledge of cyanobacterial AcOATs, and offer a useful basis for future studies on arginine metabolism and nitrogen storage in diazotrophic cyanobacteria. Full article

Ferritin, a physiological iron-storage protein, has emerged as a highly attractive platform for drug delivery owing to its biocompatibility, structural robustness, and intrinsic ability to encapsulate and protect therapeutic cargo within its hollow nanocage. Building upon previous studies that established the baseline characteristics of engineered ferritin mutants in comparison to the wild-type protein, this work specifically investigates and directly compares the thermal stability profiles of two distinct mutated variants. These variants of human H-chain ferritin, obtained through targeted site-directed mutagenesis, feature either four or six tryptophan residues per subunit, strategically positioned toward the inner cavity of the protein shell. These modifications were intended to enhance hydrophobic interactions with guest molecules while preserving the native quaternary architecture. Temperature-dependent changes in surface hydrophobicity and solvent accessibility were probed using the environment-sensitive fluorescent dye ANS, enabling a comparative assessment of the conformational behavior of the two mutants. Overall, this study highlights how targeted modulation of the internal cavity composition of ferritin can tune both its physicochemical properties and stability, providing insights relevant for the rational design of ferritin-based nanoplatforms for biomedical applications. Full article

Background: Gastric adenocarcinomas constitute a histologically and genomically heterogeneous group of cancers. The genomic classification of gastric cancers in four groups by The Cancer Genome Atlas (TCGA) has defined a framework for pathogenic discoveries. One of the groups is associated with infection by the gamma herpes virus Epstein–Barr virus (EBV) and represents a distinct subset of gastric cancers with potential therapeutic opportunities. Methods: The EBV-associated cancers from the TCGA gastric cancer cohort were analyzed to determine specific mutational and mRNA expression profiles that set these cancers apart from other gastric cancer subtypes. The cBioportal for Cancer Genomics site was used for downloading and analyzing the primary data. Results: EBV-associated cancers represented about 7% of the cohort. Mutations in the catalytic alpha subunit of PI3K kinase, PIK3CA, and the epigenetic modifiers ARID1A and BCOR were common. PIK3CA mutations were observed in 80% of EBV-associated cancers and frequently affected the hotspot codons E542 and E545. The few cases without PIK3CA mutations displayed frequent alterations in ERBB2 or in the regulatory unit of PI3K. EBV-associated cancers did not display excess cytidine to thymine (C>T) transitions compared with other gastric cancer genomic subtypes, as would be expected from the high genome methylation caused by the virus. In contrast, an increased rate of T to G (T>G) transversions was observed in EBV-associated cancers. Translesion polymerase eta (POLH), which produces a signature characterized by a preponderance of T>G, was up-regulated in EBV-associated gastric cancers and may be a contributing factor in this increase, up-regulated by wild-type p53 and over-expression of transcription factor IRF1. Conclusions: The data presented here suggest that mutagenesis in the EBV-associated gastric cancers is not a direct consequence of the virus-derived hypermethylation. Up-regulation of kinase PI3K and its pathway is a prerequisite for EBV transformation, and epigenetic alterations are frequently present, suggesting therapeutic avenues. Full article

Background: Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus–host interactions. Methods: Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Results: Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of −83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (K_D = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). Conclusions: This study not only provides novel candidate molecules for elucidating the mechanism of HS–virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs. Full article

Background: Doramectin (CHC-B1) is an excellent antiparasitic drug produced by feeding cyclohexanecarboxylic acid (CHC) to Streptomycesavermitilis bkd⁻ mutants. AveC, a bifunctional enzyme encoded by aveC (sav_0940), catalyzes the stereospecific spiroketalization and selective dehydration of dihydroxy ketone polyketide intermediates and modulates both the yield and the proportion of avermectin/doramectin in Streptomyces avermitilis. In our previous work, we constructed a strain harboring a synthetic aveC* gene encoding ten amino acid mutations, which produced nearly pure doramectin. However, the doramectin yield achieved only approximately 60% of the total doramectin and CHC-B2 output observed in the parental strain. Methods: To investigate the roles of Ser138 and Ala139 of AveC in the biosynthesis of doramectin and avermectin, site-directed mutagenesis was performed at both sites. The production and proportion of avermectin and doramectin were determined using high-performance liquid chromatography (HPLC). AlphaFold2-based molecular docking simulations were used to interpret the results. Results: Among the tested mutants, S138G, S138T, and A139H exhibited the highest doramectin production, achieving 143.87%, 151.22%, and 153.36% of the control level, respectively. Unfortunately, almost none of the tested mutants showed a positive effect on avermectin production. Molecular docking simulations revealed distinct affinities of these mutants for the dihydroxy ketone polyketide intermediate, both with and without a cyclohexyl group. Notably, all three mutants displayed larger substrate-binding cavity volumes compared with the wild-type enzyme, which likely facilitates doramectin synthesis by effectively accommodating the cyclohexyl moiety. Docking results further indicated that Ser138 and Ala139 are positioned within the binding cavity but probably do not directly participate in the dehydration activity. Conclusions: These findings suggest that optimizing cavity size through residue substitutions can enhance substrate specificity for doramectin production while preserving catalytic functionality. Full article

The Lysobacter capsici XL1 β-lytic protease (Blp) is a bacteriolytic enzyme that hydrolyzes peptide bonds in the interpeptide bridge of the peptidoglycan of Gram-positive bacteria, including antibiotic-resistant strains of pathogenic bacteria. The Blp has been extensively characterized. The only unexplored aspect is the mechanism by which this enzyme recognizes target cells. In this work, we demonstrated for the first time that the Blp structure contained a C-terminal subdomain that can be responsible for this interaction. Molecular modeling suggested a hydrophobic nature of the interaction between the Blp and peptidoglycan. Model mutant forms of the Blp, which have fewer hydrophobic areas in the C-terminal subdomain, also had fewer sites for potential interaction with the ligand. Wet lab experiments showed that these mutant Blp forms exhibited poorer binding to peptidoglycan and living Staphylococcus aureus 209P cells, resulting in decreased bacteriolytic and proteolytic activity. Amino acid residues N136 and Y160 in the C-terminal subdomain were identified and can be important for the interaction of the enzyme with target cells. Further research into the mechanism of target cell recognition by bacterial bacteriolytic proteases will enable the use of this knowledge to expand the specificity of action of these enzymes, including as antimicrobial agents for medical applications. Full article

Andes virus (ANDV), a highly pathogenic orthohantavirus, enters host cells through low pH–triggered membrane fusion mediated by the Gc glycoprotein, a class II fusion protein containing a single C-terminal transmembrane domain (TMD). While the ectodomain has been extensively characterized, the role of the TMD in late-stage fusion remains unclear. Here, we investigated the minimal functional length and sequence requirements of the ANDV Gc TMD using site-directed mutagenesis. C-terminal deletion mutants and serine-to-alanine substitutions were evaluated for protein expression, virus-like particle production, cell–cell fusion, pseudotyped vector entry, and hemifusion activity. Deletion of the Gc cytoplasmic tail (CT) or a single C-terminal TMD residue was tolerated, whereas deletion of two or more residues impaired particle production and fusion, indicating that at least 21 of the 22 TMD residues are required for efficient membrane fusion and viral entry. Hemifusion assays showed that deletion of two or three residues, or substitution of the strictly conserved S1121, allowed lipid mixing but blocked progression to full fusion, while deletion of four residues also abolished hemifusion. In contrast, mutation of the less conserved S1126 had minimal effect. These results identify a precise TMD length and a conserved polar TMD residue as critical determinants of fusion pore formation in ANDV. Full article

The orphan insulin receptor-related receptor (IRR), in contrast to its homologs from the insulin receptor family, is activated by a mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by two synergistic sites located in the dimeric extracellular domain. Here, we describe artificial mutations in the IRR transmembrane domain that promote receptor activation. First, using molecular modeling based on the NMR-derived structure, we proposed amino acid substitutions that could enhance non-covalent interactions between the transmembrane segments of the IRR dimer. These mutations were subsequently tested for effects on pH sensing by IRR. We showed that double-mutant A938E-A939R was highly phosphorylated at neutral pH and still sensitive to alkaline pH. Remarkably, the double substitution of V929E-G930R resulted in strong basal phosphorylation of the receptor over the pH titration range. Through site-directed mutagenesis, we demonstrated that the transmembrane domain plays a critical role in IRR activation, allowing for targeted control of functioning of the receptor, including its pH sensitivity. Full article

Endophytic fungi are promising sources of novel antiviral compounds, and the crude extract from Alternaria alternata PO4PR2 has previously shown anti-HIV-1 activity. This study evaluated its efficacy against integrase strand-transfer inhibitor (INSTI)-resistant HIV-1 and its mechanism of action. Key resistance mutations (Y143H, G118R, N155H, and R263K) were introduced into the HIV-1 pNL4.3 clone via site-directed mutagenesis and confirmed through Sanger sequencing. Viral infectivity was assessed in TZM-bl cells, while cytotoxicity was measured using an MTT assay. Antiviral activity was determined through a luciferase-based assay, and integration inhibition was evaluated using integrase activity assays and Alu-gag nested PCR. The extract demonstrated potent inhibition of resistant mutants, with low IC₅₀ values (0.02971–0.1652 μg/mL), and showed minimal cytotoxicity (CC₅₀ = 300 μg/mL), maintaining over 80% cell viability. It inhibited integrase activity by 67%, specifically targeting the strand-transfer step, and significantly reduced integrated viral DNA. Molecular docking of 14 compounds identified coumarin derivatives as key bioactive metabolites, exhibiting mutation-tolerant binding within the integrase catalytic pocket. Overall, these findings highlight PO4PR2 as a promising source of compounds for developing new therapies targeting drug-resistant HIV-1 integrase. Full article

Endogenous estrogens are implicated in carcinogenesis through both estrogen receptor-mediated cell proliferation and the direct genotoxicity of reactive metabolites. Oxidative metabolism of estrogens produces catechol estrogens that are further converted to electrophilic ortho-quinones capable of alkylating DNA. The prevailing model of mutagenesis proposes that these N3Ade and N7Gua adducts depurinate to form abasic sites that induce mutations initiating hormone-related cancers. However, the mutation spectrum observed in experimental data is inconsistent with this mechanism, and synthetic studies of estrogen-DNA adducts have relied on acidic conditions that artificially promote depurination, leaving stable N7-dG lesions poorly understood. To address this, we synthesized stable N7-dG catechol and estrone adducts using 2′-fluorinated deoxyguanosine, a modification that inhibits N-glycosidic bond cleavage. ROESY 2D NMR spectroscopy revealed through-space correlations consistent with a preferred anti-conformation in solution, supported by molecular modeling. Structural analysis suggests that these cationic aryl adducts likely preserve the Watson–Crick base pairing edge but may promote tautomerization capable of altering base pairing and generating G-to-A mutations. These findings provide the first synthesized stable models of N7-dG estrogen adducts and may support an alternative mechanism of estrogen-induced mutagenesis independent of depurination, enabling future biochemical investigations of related DNA repair and mutagenesis. Full article

Bst DNA polymerase is a biotechnologically modified thermostable enzyme from the thermophilic Gram-positive bacterium Geobacillus stearothermophilus. The unique structure of Bst DNA polymerase determines its thermal stability, ability to replace a DNA strand and specificity. The high specificity of Bst DNA polymerase ensures the efficiency, sensitivity, and high rate of loop-mediated isothermal amplification (LAMP), which is widely used in vitro biotechnology. The review reveals the structural and functional features of the enzyme, its application in LAMP and methods of improvement of thermal stability (including directed evolution, site-directed mutagenesis, fusion constructs, and chemical modifications). The terminal transferase activity and ab initio synthesis are discussed regarding problems of Bst DNA polymerase and the ways to eliminate them. The questions of introducing modified nucleotides and primers to expand the diagnostic capabilities of LAMP are also discussed. Modern advances in Bst DNA polymerase engineering pave the way for the creation of reliable, thermostable, and highly specific test systems suitable for widespread diagnostic applications. Full article

Escherichia coli, a facultative anaerobic Gram-negative member of the Enterobacteriaceae, is an increasingly important opportunistic pathogen driven in part by rising resistance to clinically important antibiotics. Regulation of multidrug efflux systems by two-component signal transduction pathways, particularly the BaeSR system, plays a central role in this process. However, the functional residues governing signal transduction through the sensor kinase BaeS remain incompletely defined. In this study, we integrated domain prediction, homology-guided site-directed mutagenesis, in vitro protein purification, autophosphorylation assays, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)-based transcriptional analysis of selected BaeSR-regulated genes to delineate key residues required for BaeS function. Sequence analysis identified His250 as a candidate autophosphorylation site and Asn364 as a conserved residue within the catalytic domain. Biochemical characterization of purified wild-type BaeS and an H250A mutant demonstrated that His250 is indispensable for autophosphorylation. Consistently, RT-qPCR analysis showed that BaeS activation markedly induced the transcription of BaeSR-regulated efflux-associated genes, whereas genetic deletion of baeS or selective disruption of kinase activity by the N364A mutation abolished this response. Together, these findings establish His250 as a key residue for BaeS autophosphorylation and identify Asn364 as essential for inducible BaeSR signaling and activation of resistance-associated target genes, thereby establishing an experimental framework for elucidating BaeSR-mediated efflux regulation and informing future studies of resistance regulatory networks and potential intervention strategies centered on key signaling nodes. Full article

Streptococcus parauberis is a major pathogen responsible for streptococcosis in both marine and freshwater fish species, causing substantial economic losses in aquaculture. The increasing prevalence of multidrug resistance has highlighted the urgent need for alternative disease control strategies. Interference with bacterial quorum sensing (QS) systems represents a promising approach. This study aimed to identify and biochemically characterize an Rgg-family transcriptional regulator and evaluate its potential as a target for quorum sensing-related regulatory interference in vitro. We hypothesized that this Rgg regulator may function as a quorum sensing-associated transcription factor capable of promoter binding and modulation by small molecules. Bioinformatic analyses were used to identify the rgg gene encoding an Rgg-family transcriptional regulator and predict its structural features. The gene was cloned, heterologously expressed, and purified. Promoter binding activity was examined using electrophoretic mobility shift assay (EMSA), and key amino acid residues were identified through site-directed mutagenesis. The inhibitory effect of the cyclic peptide cyclosporin A (CsA) on Rgg-promoter binding was further assessed. The rgg gene (864 bp) encoding a 287-amino-acid protein (34.1 kDa) was successfully identified and expressed. Purified Rgg specifically bound to its own promoter region in a concentration-dependent manner. Mutations at conserved arginine residues R12 and R15 within the helix-turn-helix DNA-binding domain abolished promoter binding activity. Furthermore, CsA disturbed Rgg-promoter binding in a dose-dependent manner. This study provides the first in vitro characterization of an Rgg-family transcriptional regulator in fish-derived S. parauberis. The findings expand current understanding of Rgg-family regulators potentially associated with quorum sensing in aquatic streptococci and provide a preliminary basis for further investigation of quorum sensing-related regulatory interference strategies for controlling streptococcal diseases in aquaculture. Full article