Nam-Chul Ha

Morpholino nucleosides play an important role in medicine as building blocks for antisense oligonucleotide drugs (PMOs), but their therapeutic applications as nucleosides are still unexplored. A novel library of 38 fused bicyclic morpholino-uridine and -adenosine derivatives was constructed through an innovative, single-step strategy based on a reductive aminocyclization reaction between readily prepared nucleoside secodialdehydes and bifunctional amines. By fine-tuning the reaction conditions, a diverse array of morpholino bicycles was obtained with high/complete stereoselectivity. With aminothiols and diamines, the NaCNBH3–AcOH reducing agent resulted in 3′-fused or 5′-fused ring systems, depending on the amine reactant, while the NaCNBH3–ZnCl2-mediated reduction gave predominantly 5′-fused bicycles due to intramolecular 3′-hydride donation by the reducing agent coordinated to the nucleobase. All new compounds were evaluated for their antiviral activity against SARS-CoV-2. Three analogues demonstrated significant efficacy, exhibiting single-digit micromolar EC50 values. Mechanistic investigations revealed that these compounds inhibit the viral life cycle at a postentry stage, with one compound potentially affecting later stages of the viral replication cycle. Furthermore, molecular docking, enzymatic inhibition studies, and in silico ADMET analyses were conducted. The findings emphasize the potential of this fused bicyclic system as an attractive scaffold in nucleoside chemistry and advocate for its further optimization as a promising class of SARS-CoV-2 inhibitors.

Highlights: • LLM-driven ecological priors enable large-scale functional labeling of CGL homologs. • EnzFormer integrates ESMC embeddings to accurately predict putative enzyme activity. • In silico screening identifies a distal V129G variant with a 2-fold higher k _cat . • V129G illustrates a stability–activity trade-off via active site loop flexibility. • Specialized AI models reveal nonobvious allosteric sites modulating enzyme dynamics.

The diversity of protein structures and reaction mechanisms complicates general-purpose AI models for enzyme engineering, motivating family-specialized models. In this study, we developed EnzFormer, a specialized AI pipeline for engineering Staphylococcus aureus L-cystathionine gamma-lyase (SaMccB). To overcome the scarcity of experimental labels, we used GPT-4o to generate putative activity labels for CGL homologs, leveraging species-level ecological and evolutionary metadata as a proxy for functional selection. Using these labels, we trained a Transformer classifier on ESMC protein language model embeddings. From an exhaustive single-mutant library, in silico prioritization nominated four variants for testing and identified SaMccB V129G with an ~2-fold increase in kcat relative to wild-type. Remarkably, Val129 is distal to the active site; however, crystallographic and biochemical analyses suggest that the V129G weakens local packing, thereby increasing the conformational flexibility of the active-site loop (residues 95–103), consistent with faster conformational steps in the catalytic cycle. Together, these results indicate that combining LLM-derived evolutionary priors with a family-specific predictive model can pinpoint non-obvious distal mutations that tune enzyme dynamics, providing a scalable strategy for engineering PLP-dependent enzymes.

The Bacillus subtilis spore crust is an exceptionally robust proteinaceous layer that protects spores under extreme environmental conditions. Among its key components, CgeA, a glycosylation-associated protein, plays a critical role in modifying crust properties through its glycosylated moiety, enhancing spore dispersal in aqueous environments. In this study, we present the high-resolution cryo-electron microscopy structure of the core region of CgeA at 3.05 Å resolution, revealing a doughnut-like hexameric assembly. The N-terminal regions are disordered, whereas the C-terminal region forms the core of the hexamer. Although the loop containing Thr112 was not resolved in the density map, its location can be inferred from surrounding residues, suggesting that Thr112 is situated on the exposed surface of the hexamer. On the opposite face, a distinct electrostatic pattern is observed, featuring a negatively charged central pore and a positively charged outer surface. Modeling and biochemical studies with the putative glycosyltransferase CgeB provide insights into how the glycosyl group is transferred to Thr112. This study offers a molecular-level understanding of the assembly, glycosylation, and environmental adaptability of the B. subtilis spore crust, with valuable implications for controlling spore formation in industrial applications.

In eukaryotic cells, mitochondria and the endoplasmic reticulum (ER) form close contacts at mitochondria-associated ER membranes (MAMs), which are involved in diverse cellular processes. The outer mitochondrial membrane protein Fis1, known for its role in mitochondrial fission, has been reported to interact with the ER-resident protein Bap31. Here, we present crystal structures of the cytosolic domain of human Fis1 in two distinct conformations, along with a co-crystal structure of Fis1 bound to the C-terminal region of the Bap31_vDED domain. One Fis1 structure resembles monomeric yeast Fis1 and features a characteristic N-terminal “Fis1 arm” conformation, which may indicate an autoinhibitory function. In the co-complex, the Bap31_vDED region engages the convex surface of Fis1’s tetratricopeptide repeat (TPR) domain. These findings provide structural insight into the interaction between Fis1 and Bap31 at ER–mitochondria contact sites.

Cronobacter sakazakii is a Gram-negative pathogen that causes severe infections such as neonatal meningitis and sepsis. Bacteriophages (phages) rely on tail fibers for host recognition and infection, but the mechanisms of how phages recognize their bacterial hosts are not completely elucidated. In this study, two lytic C. sakazakii phages belonging to the Drexlerviridae family, CRES7 and CRES9, were isolated from sewage in South Korea. The genomes of both phages are almost the same, with only two nucleotide differences in the gene encoding a putative tail fiber, causing two amino acid differences at amino acid residues 400 and 550 of the tail fiber. The predicted structure of the tail fiber revealed that the two amino acid residues are located on the surface of the tail fiber, suggesting that these two amino acid residues may affect receptor binding. These amino acid differences resulted in differential host ranges, adsorption rates, and burst sizes of CRES7 and CRES9; CRES7, which could infect only the C. sakazakii serotype O1 strain, exhibited a higher adsorption rate and larger burst size compared to CRES9, whereas CRES9 could infect both serotypes O1 and O3 strains. These findings provide insights into how the mutations in the tail fiber gene contribute to the fitness of phages within natural environments and help develop phage-based strategies with expanded host range or enhanced specificity for targeted biocontrol of C. sakazakii . IMPORTANCE: Accurate recognition and attachment to the bacterial host, mediated by tail fibers, are crucial for successful phage infection. Understanding the mechanisms underlying host specificity of phages is essential for developing targeted biocontrol applications. This study identified specific amino acid residues responsible for host specificity in the tail fibers of two newly isolated Cronobacter sakazakii phages, CRES7 and CRES9. Differences in these residues showed variation in O serotype recognition, leading to differences in host range, adsorption efficiency, and burst size. These findings provide valuable insights into tail fiber-mediated host specificity, facilitating the development of more effective phage-based strategies against C. sakazakii .

Bacillus cereus, a Gram-positive aerobic bacterium commonly found in soil, food, and water, forms endospores that can withstand harsh environmental conditions. The endospores are encased in a protective spore coat consisting of multiple layers of proteins, among which, CotE serves as a crucial morphogenetic protein within the outer coat. In this study, we observed that the homotrimeric CotE protein underwent further oligomerization induced by Ca ²⁺ and was subsequently dissociated by dipicolinic acid, a compound released from the spore core during germination. Through cryo-electron microscopy and tomography analyses of the Ca ²⁺ -induced CotE oligomer, combined with structural predictions and biochemical studies, we propose a three-dimensional meshwork organization facilitated by tryptophan-based interactions between CotE trimers. The resulting meshwork was organized in a defective diamond-like tetrahedral configuration. These insights enhance our understanding of how CotE contributes to endospore morphogenesis and germination through the rapid disassembly of these layers. IMPORTANCE: Bacterial endospores are highly resilient structures that allow bacteria to survive extreme environmental conditions, making them a significant concern in food safety and healthcare. The protein CotE plays a critical role in forming the protective outer coat of these endospores. Our research uncovers the three-dimensional meshwork architecture of CotE and reveals how it contributes to the structural integrity and rapid disassembly of endospores during germination. By understanding CotE’s unique 3D structure and its interaction with other molecules, we gain valuable insights into how bacterial endospores are formed and how they can be effectively targeted for sterilization. This work not only advances our fundamental knowledge of bacterial endospore biology but also has potential applications in developing new strategies to combat bacterial contamination and improve sterilization techniques in the food and healthcare industries.