Yanwei Li

Human ornithine aminotransferase (hOAT) is overexpressed in hepatocellular carcinoma (HCC) and participates in glutamine anabolic metabolism, making it a promising therapeutic target. Although (1S,3S)-3-amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (named here as BCF₃ after the (bis)trifluoromethyl feature) has been proposed as a potent inhibitor of hOAT, previous experimental studies have characterized an external aldimine intermediate and the static end-state adduct, but the elementary fluorine-elimination steps and their modulation by the active-site environment remain incompletely understood. Herein, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to systematically explore the irreversible inhibitory mechanism mediated by external aldimine II—the key reaction intermediate of BCF₃—against hOAT. Conserved active-site water molecules act as proton-shuttling bridges, and residue-dependent electrostatic effects control fluorine elimination. External aldimine II forms a stable covalent adduct with Lys292 and sequentially releases fluorine atoms from the –CF₂ moiety via water-mediated F^α− and F^β− elimination, with feasible barriers of 10.8 and 16.0 kcal/mol, respectively. Negative overall reaction energies indicate that the elimination products are energetically favored in the system. Electrostatic and noncovalent interaction analyses suggest that Arg180 and Gly42 promote fluorine elimination whereas Phe177 suppresses it, providing an atomistic explanation for the superior inhibitory potency of the bis-trifluoromethyl scaffold. The atomistic inhibitory mechanism uncovered in this study provides a theoretical foundation for the future rational design of hOAT-targeted drugs for hepatocellular carcinoma therapy.

The exploration of unique metallophilic interactions, a pivotal category of intermolecular interactions that govern material properties and reactivity, is crucial for deciphering the role of metal atomic orbitals in their formation and uncovering their fundamental characteristics. However, due to their inherently subtle and delicate nature, these interactions remain exceptionally challenging to detect and characterize. Here, we present a method of utilizing metallophilic interactions to build stable graphene–molecule–graphene single-molecule junctions through molecular engineering. Real-time electrical measurements reveal that the metal atomic orbital contributions in various metal–metal interactions originate from the hybridization of different angular components of intermetallic d and s orbitals (dz2–s/dxy–s), a conclusion further corroborated by theoretical calculations. Distinct overlaps between intermetallic orbitals give rise to diverse molecular configurations, leading to corresponding orbital-resolved conductance. Furthermore, we establish the relationship between the orbital interaction and experimental transition activation energy through counterion- and metal-dependent measurements. This work uncovers the underlying mechanism of metal–metal interactions, bridges the gap between covalent bonds and weak interactions, and extends the research scope of molecular electronics.

Enzymatic depolymerization of hydrolyzable plastics, such as polyurethane (PU), opens a promising route for the eco-friendly recycling of plastic waste. A comprehensive understanding of the molecular mechanisms governing enzyme-catalyzed PU hydrolysis is crucial for engineering high-performance enzymes. Here, we combined extensive molecular dynamics simulations, hybrid quantum mechanics/molecular mechanics calculations, and experimental mutagenesis to elucidate how residues in distinct spatial regions regulate substrate binding and catalysis in UMG-SP2. Our results reveal that residues in the first shell (e.g., L140) stabilize the substrate in a catalytically productive conformation within the active-site pocket. Three flexible loop regions, H215–L227, L320–D346, and S377–L400, are identified as potential regulators of substrate capture. Furthermore, we quantify the contributions of individual surface-exposed charged residues to the reaction-relevant electric fields in the active site. The reaction mechanism comprises acylation and deacylation stages, with the rate-determining step corresponding to water-assisted nucleophilic attack. Experimental mutagenesis demonstrates that substitutions at residues in different spatial regions selectively modulate substrate binding and catalytic activity, leading to distinct and substrate-dependent effects on low-molecular-weight dicarbamate hydrolysis and PU depolymerization. Collectively, these findings reveal a residue-position-dependent modulation framework for urethanase activity and establish a general mechanism-guided strategy for advancing enzymatic PU depolymerization and recycling.

Current research on interfacial enzyme catalysis of starch granules largely overlooks interactions with molecules of specific spatial conformations. Here, we demonstrated that the CBM20-truncated mutant AmyM-TR2 exhibited over 30-fold greater hydrolysis efficiency toward starch granules compared to wild-type AmyM, with a shifted preference toward amylopectin. Molecular docking revealed that the exposed domain C in AmyM-TR2 enables binding to double-helical α-glucan molecules by three key residues (R351, W374, and W392). Interfacial catalytic analysis revealed that the discrepancy of AmyM and AmyM-TR2 in hydrolysis efficiency toward starch granules was related to substrate-targeting specificity and corresponding targeted productive binding sites (PBSs). AmyM-TR2 demonstrated a superior sustained catalytic capacity toward its targeted PBSs within multi-component starch. The processive catalytic capacity of AmyM-TR2 toward amylopectin double helices in starch granules is the fundamental determinant of its highly efficient degradation, providing new insights for interfacial catalytic mechanisms and modification of interface enzymes. IMPORTANCE: Interfacial enzyme reactions are ubiquitous in both natural processes and industrial applications. Understanding the mechanistic aspects of enzymatic action on insoluble substrates is crucial for advancing sustainable biomass conversion and waste treatment technologies. Starch granule degradation mediated by raw starch-digesting enzymes (RSDEs) is a classical interfacial enzymatic reaction. The mechanism of RSDEs on starch granules is attractive, as it effectively circumvents the high energy consumption and viscosity challenges associated with conventional high-concentration starch hydrolysis processes. In this study, we demonstrate interfacial catalysis in starch granule depolymerization by substrate-targeting modification in raw starch-degrading amylase AmyM by exposing novel starch-binding modules, thus establishing a critical link between catalytic characteristics of amylase and starch substrate properties, which provides new insight for the contributions to insoluble substrate degradation within heterogeneous systems.

Polycyclic aromatic hydrocarbons (PAHs) and their nitrogen-containing derivatives are persistent environmental pollutants prevalent in combustion byproducts and industrial waste. Human cytochrome P4501A1 (CYP1A1), a key isoform within the cytochrome P450 enzyme family, plays a pivotal role in its oxidative metabolism. Here, a high-level quantum mechanics/molecular mechanics (QM/MM) computational approach was employed to elucidate the regioselective activation mechanisms of three representative compounds: carbazole, fluorene, and 2-aminofluorene. We show that CYP1A1 preferentially activates these compounds through competitive electrophilic addition and hydrogen abstraction pathways. We identify different regioselectivities among the three model compounds. Correlation analyses reveal that distance D_O-C, D_O-H, and angle A_C-O-Fe are the principal determinants of regioselectivity. The calculated reactivity order is 2-aminofluorene > carbazole > fluorene. These findings provide mechanistic insight into the enzymatic transformation of PAHs and their nitrogen-containing derivatives in human cells, contributing to a more accurate assessment of their potential health risks.

Enzymatic recycling of polyethylene terephthalate (PET) has been recognized as an eco-friendly option for addressing the global plastic waste problem. Fully deciphering the catalytic mechanism is vital for designing high-performance enzymes. Here, we performed quantum mechanics/molecular mechanics molecular dynamics simulations to systematically explore the depolymerization mechanism of PET by the hydrolase LCC ^ICCG . We demonstrate that both PET chain binding and product release require free energy barriers, whereas the rate-determining step corresponds to a catalytic process with a free energy barrier of 20.4 kcal·mol ^-1 . We also observe that the enzyme internal electric field varies dynamically throughout the catalytic process. Oriented external electric field analysis indicates that this “dynamic shift” stabilizes the transition state more than the reactant, thereby lowering the energy barrier. We anticipate that these insights will contribute to the rational engineering of PET hydrolases by optimizing their dynamic internal electric fields.