Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H₂S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H₂S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5′-phosphate-binding site. Gallic acid has little effect on the other H₂S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.

Thymidylate synthase (TYMS) is an essential enzyme for the de novo synthesis of deoxythymidine monophosphate (dTMP) and has been a primary target for cancer chemotherapy. Although the physical structure of TYMS and the molecular mechanisms of TYMS catalyzing the conversion of deoxyuridine monophosphate (dUMP) to dTMP have been the subject of thorough studies, its oligomeric structure remains unclear. Here, we show that human TYMS not only exists in dimer form but also as an octamer by intermolecular Cys43-disulfide formation. We optimized the expression conditions of recombinant human TYMS using the Escherichia coli system. Using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), we have shown that purified TYMS has catalytic activity for producing dTMP. In the absence of reductant β-mercaptoethanol, SDS-PAGE and size exclusion chromatography (SEC) showed that the size of the TYMS protein is about 35 kDa, 70 kDa, and 280 kDa. When the Cys43 was mutated to Gly, the band of ~280 kDa and the peak of the octamer disappeared. Therefore, TYMS was determined to form an octamer, depending on the presence of Cys43-disulfide. By measuring steady-state parameters for the monomer, dimer, and octamer, we found the k_cat of the octamer was increased slightly more than the monomer. On the basis of these findings, we suggest that the octamer in the active state might have a potential influence on the design of new drug targets.

Antibiotic resistance is a critical global health threat, already causing over 1.27 million deaths annually and projected to exceed 10 million by 2050. This crisis is compounded by stagnation in novel antibiotic discovery, highlighting the need for mechanism–based innovation. Here, we provide an integrative framework linking antibiotic mechanisms of action, bacterial resistance pathways, and emerging therapeutic strategies. Antibiotics are systematically categorized by their molecular targets, cell wall synthesis, membrane integrity, nucleic acid replication, protein synthesis, and metabolic pathways, while resistance mechanisms are outlined in parallel, including enzymatic degradation, target modification, efflux, and permeability barriers. We further highlight novel approaches such as structure–guided drug design, synergistic combinations, nanoparticle delivery, and artificial intelligence–driven discovery. Precision medicine and microbiome modulation are also emphasized as next–generation interventions. By bridging molecular mechanisms with translational strategies, this review outlines opportunities to guide antibiotic innovation and advance precision therapies against the escalating threat of antimicrobial resistance.

Klebsiella pneumoniae is the second-most common opportunistic pathogen in clinical practice and has developed resistance to potent antibacterial drugs such as carbapenems. Therefore, developing safe and effective strategies for the prevention and treatment of K. pneumoniae infections remains a critical challenge. In this study, a strain named Tie-10 isolated from Antarctic samples demonstrated potent antibacterial activity against K. pneumoniae, which was subsequently identified as Bacillus nakamurai. The fermentation medium and culture conditions were systematically optimized through single-factor experiments, orthogonal array testing, and response surface methodology. The optimal medium composition was determined to be beef extract, peptone, and KNO₃. The culture conditions included a time of 24 h, temperature of 37 °C, pH of 7.0, and bottling volume of 80 mL. Antagonistic experiments demonstrated that the crude extract of B. nakamurai Tie-10 exhibited significant inhibitory activity against K. pneumoniae. The alkaline protease (AKP) assay demonstrated that the crude extract effectively disrupted the cellular integrity of K. pneumoniae, a finding further corroborated by scanning electron microscopy (SEM) analysis. Furthermore, the crude extract significantly inhibited extracellular protease secretion in K. pneumoniae, downregulated the expression of virulence-associated genes, and effectively disrupted biofilm formation. The study presented innovative strategies for the management and containment of K. pneumoniae infections.