Improving enzyme stability without compromising catalytic activity remains a major challenge in protein engineering. Here, we present a co-evolution-guided strategy to enhance both thermostability and catalytic performance of the flavin-dependent monooxygenase MonCI, an enzyme involved in monensin biosynthesis. By combining sequence covariation analysis with structural filtering, a focused library of 15 single mutants yielded 4 variants with increased stability and activity. Combinatorial assembly generated triple, quadruple and quintuple mutants, with the best-performing quadruple variants exhibiting up to a 10 °C increase in melting temperature, a 2.3-fold increase in specific activity, and a 2.1-fold longer half-life, accompanied by enhanced turnover despite reduced substrate affinity. Crystal structures and molecular dynamics simulations reveal that stabilization arises from strengthened intramolecular networks of hydrogen bonds, salt bridges, and hydrophobic interactions, while epistatic effects limit additive improvements. This work provides mechanistic insight into how co-evolving residues modulate enzyme structure and function, presents a useful co-evolution-guided strategy for enzyme design, and advances MonCI as a promising biocatalyst for asymmetric epoxidation.

Genetic mutations frequently disrupt protein structure, stability, and solubility, acting as primary drivers for a wide spectrum of diseases. Despite the critical importance of these molecular alterations, existing computational models often lack interpretability and fail to integrate essential physicochemical interactions. To overcome these limitations, we propose SheafLapNet, a predictive framework grounded in the mathematical theory of Topological Deep Learning (TDL) and Persistent Sheaf Laplacian (PSL). Unlike standard Topological Data Analysis (TDA) tools such as persistent homology, which are often insensitive to heterogeneous information, PSL explicitly encodes specific physical and chemical information such as partial charges directly into the topological analysis. SheafLapNet synergizes these sheaf-theoretic invariants with advanced protein transformer features and auxiliary physical descriptors to capture intrinsic molecular interactions in a multiscale and mechanistic manner. To validate our framework, we employ rigorous benchmarks for both regression and classification tasks. For stability prediction, we utilize the comprehensive S2648 dataset, alongside the independent S350 and strictly non-redundant S669 blind test sets to ensure robust evaluation and thermodynamic consistency. For solubility prediction, we employ the PON-Sol2 dataset, which provides annotations for increased, decreased, or neutral solubility changes. By integrating these multi-perspective features, SheafLapNet achieves state-of-the-art performance across these diverse benchmarks, demonstrating that sheaf-theoretic modeling significantly enhances both interpretability and generalizability in predicting mutation-induced structural and functional changes.

A uni-molecular probe capable of guiding tumor detection, resection, and postoperative healing monitoring remains an unmet need in precision medicine. HClO, with well-defined dualistic roles in tumor progression and tissue repair, offers an ideal biomarker to bridge these processes, but existing probes are limited by intrinsic background fluorescence, instrument-dependent signal variability, and an inability to function across this clinical workflow. Here we report Cy5Ql-HClO, a near-infrared activatable probe employing a dual photoinduced electron transfer (PET) quenching strategy to achieve true zero-intrinsic fluorescence. By integrating an electron-deficient quinolinium at the Cy5 meso-position (primary PET channel) with a dimethylthiocarbamate recognition unit (secondary PET quencher), the probe remains completely dark until HClO-specific cleavage triggers a self-eliminating cascade, restoring strong fluorescence. This design yields undetectable background quantum yield (<0.001), 26 nM sensitivity, and instrument-independent signal quantification across platforms. In tumor-bearing mice, Cy5Ql-HClO enabled high-contrast visualization of endogenous HClO in tumors, guiding resection with sub-2 mm margins (histologically confirmed). Postoperatively, longitudinal imaging captured a distinct HClO burst on day 2 that decreased sharply by day 3, corroborated by western blot and immunohistochemical analysis of myeloperoxidase (MPO) and the neutrophil marker Ly6G. By uniting tumor resection guidance with postoperative monitoring within a single probe, Cy5Ql-HClO provides a versatile tool for investigating HClO biology across interconnected pathological and reparative processes.

The majority of human senescent cells exhibit overexpression of senescence-associated β-galactosidase (SA-β-gal), rendering this enzyme the most extensively utilized biomarker of senescence and a key prodrug target for intervening in aging. However, strategies for the authentic in situ identification of SA-β-gal with single-molecule resolution have not been established, limiting the accuracy of senescence targeting. Here we present an unprecedented molecular approach to identify SA-β-gal in a manner that avoids dissociation from the target enzyme, which integrates the atomic hybridization of SA-β-gal substrates with protein-environment-sensitive fluorescence technology. Using this design principle, we created nine fluorescent probes with distinct working modes for the single-molecule (target enzyme)-resolved identification of SA-β-gal. Five of them formed crystal complexes with the wild-type β-galactosidase by binding at the active site, clearly evidencing their single-molecule resolution capability. Our strategy, capable of both identifying and locating SA-β-gal, facilitated its application in dynamic single-molecule localization microscopic imaging, achieving the in situ tracking of SA-β-gal in living cells at the nanoscale. Significantly, the probing of SA-β-gal with high fidelity enabled the precise evaluation of the aging degree in mice. As such, our research provides a promising method for the authentic in situ identification of this senescence-associated protein with single-molecule resolution.

Polyether polyketides are a structurally diverse group of natural products known for their antimicrobial and antiproliferative activities. Lasalocid A is a canonical natural polyether produced by the soil bacterium Streptomyces lasalocidi. In lasalocid A biosynthesis, a polyene polyketide intermediate is converted into a bisepoxide by the flavin-dependent monooxygenase enzyme Lsd18. Remarkably, Lsd18 acts on two distinct C═C groups in the substrate molecule, forming two (R,R) epoxides. We have determined the X-ray crystal structures of Lsd18 in the substrate-free, substrate-bound, and product-bound forms. Our work has revealed that Lsd18 has an extra-large substrate-binding pocket that allows the polyene to adopt different conformations within the enzyme pocket. This feature enables Lsd18 to epoxidate both of the C═C groups. Additionally, a subpocket located near the Lsd18 active site controls stereoselectivity by dictating which face of the C═C group is placed next to the flavin. Molecular understanding of how Lsd18 transforms a polyene into a bisepoxide during lasalocid A biosynthesis lays the foundation for the production of designer polyethers for drug development.

Fluorescence imaging-guided photodynamic therapy (PDT) offers immense clinical potential for cancer treatment. However, their therapeutic efficacy and biosafety are compromised by the high oxygen dependency of traditional Type II photosensitizers and insufficient targeting accuracy. Herein, we present a tumor-organelle-targeted and activatable phototheranostic platform (termed NO2/BDP-BT), which was engineered via an atom-economical all-in-one design strategy. The NO2/BDP-BT gathers exceptional tumor-organelle targeting ability, nitroreductase (NTR)-activated fluorescence enhancement, allowing it to monitor hypoxia levels in biosystems (living cells, clinical patient tissues, and in vivo). Moreover, it enables the generation of Type I/II reactive oxygen species (ROS) in situ after NTR activation, thereby suppressing tumor growth with an inhibition rate of 93.2 % via mitochondria-mediated apoptosis and potentiating the antitumor immunity response. Cocrystal structural analysis of the NTR protein in complex with the precursor of NO2/BDP-BT (PDB: 7XWW; resolution: 2.80 Å) first reveals that multiple non-covalent interactions (e.g., hydrogen bonding and π-π stacking) make it anchor in the catalytic environment with high affinity. Moreover, NO2/BDP-BT can be expanded to a Type I PDT photosensitizer (NO2/BDPS-BT) by introducing a thiophene unit, conquering hypoxia restriction in PDT. This work establishes a molecular platform of activatable phototheranostic sensor with potent therapeutic efficacy and biosafety, which would effectively address both hypoxia resistance and targeting deficiencies inherent in conventional PDT.

Aberrant peroxynitrite (ONOO-) may damage a wide array of biomolecules, causing cellular dysfunctions that are closely associated with multiple oxidative stress-related diseases. Therefore, detecting ONOO-levels in physiological and pathological processes is of great significance. With this research, a coumarin/phenanthridinefused probe 1 was synthesized in high yield, and exhibited high selectivity toward ONOO-for both fluorescence and UV-vis channels in DMSO/HEPES (v/v, 4/6, pH=7.4) solution. The probe exhibited a near-infrared (NIR) emission at 710 nm with a large Stokes shift of 200 nm, which was selectively quenched by adding ONOO-. The probe was used to track ONOO-in live cells, Arabidopsis thaliana, zebrafish, and mice.

Enzyme compartmentalization is a ubiquitous biochemical mechanism that nature uses to perform simultaneous but chemically incompatible metabolic processes in physically separated environments. However, it is a substantial challenge in homogeneous catalysis to spatially confine specific reaction components to prevent undesired pathways. Here we exploit the concept of compartmentalized enantioselective energy transfer catalysis by integrating artificial triplet photoenzymes and tailored triplet quenchers. The confined protein cavity was genetically encoded with a photosensitizer for enantioselective [2 + 2] photocycloaddition of 1-naphthol derivatives, while the outer bulk solution was modified with strategically introduced quenchers to inhibit the racemic background reaction induced by direct excitation, a fundamental challenge in asymmetric photocatalysis. This study not only expands the repertoire of artificial photoenzymes but also introduces a distinctive biocatalytic approach for precisely controlling reaction processes with spatial resolution, a capability that is usually unattainable in traditional chemocatalysis. Artificial photobiocatalytic reactions are appealing but sometimes suffer from non-enzymatic side reactions. Now a photoenzyme for enantioselective [2 + 2] photocycloaddition of 2-naphthyl derivatives is reported and combined with designed quenchers that shut down the competing enzyme-free racemic reaction.

Hydrogels characterized by a three-dimensional polymer network are promising vehicles for oral colon-targeted drug delivery, yet conventional systems relying on intragastric cross-linking agents face clinical limitations. Here, we present a cross-linker-independent oral in situ hydrogel (CCS/CMC-Na) functionalized with Ti₃C₂ MXene nanosheets for optimized inflammatory bowel disease (IBD) therapy. The CCS/CMC-Na hydrogel exhibits a liquid-gel state transition in simulated gastric fluids while gradually returning to a liquid state in simulated colonic fluids, enabling precise targeting of inflamed colon tissues and protecting encapsulated drugs from gastric degradation. The integration of Ti₃C₂ not only enhances mechanical stability but also introduces multifunctional therapeutic capabilities: repairing all levels of intestinal barriers, reducing oxidative stress, inhibiting apoptosis, modulating Regulatory T cells (Treg)/T helper 17 (Th17) cell differentiation and NF-κB/IκB inflammatory pathway, and remodeling the intestinal microbiota. In murine models of acute and chronic colitis, Ti₃C₂@CCS/CMC-Na significantly alleviates disease severity, outperforming clinical first-line drug 5-ASA. Crucially, this system eliminates the need for dual-tube administration, simplifies dosing protocols, and demonstrates excellent biocompatibility over 50 days, addressing key challenges in current IBD therapies. By integrating precise targeting, multimodal therapeutic actions, and scalable fabrication, Ti₃C₂@CCS/CMC-Na represents great clinical translational potential, offering a safer and more effective treatment option for IBD.

Monensin, a polyether antibiotic produced by Streptomyces cinnamonensis, is synthesized through a series of stereospecific epoxidations orchestrated by the FAD-dependent monooxygenase MonCI. This study presents the crystal structure of MonCI in complex with a substrate analog, farnesyl acetate, which undergoes in vitro epoxidation at all three olefinic sites by MonCI. Additionally, we have solved the crystal structures of MonCI in complex with mono- and bis-epoxide intermediates. Structural analysis revealed that the interactions between MonCI and its substrate are predominantly hydrophobic, with residue Y208 playing a crucial role in substrate translocation. Computational modeling emphasizes the importance of FAD proximity to each olefinic group for efficient catalysis, providing insight into the mechanistic pathways for the first, second, and third epoxidations. Notably, when combined with electrochemical techniques, the yield of tri-epoxide farnesyl acetate reached 94 %, surpassing traditional enzymatic catalysis. This work not only elucidates the catalytic mechanism of MonCI but also underscores its potential as an effective biocatalyst for processive epoxidation in organic synthesis.

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Collagen, with its most characteristic structural unit, the triple helix, has wide applications in pharmaceutical and cosmetic industries. However, the application of collagen is limited by its poor thermal stability, especially fish collagen. Fish collagen, despite its abundance and eco-friendly sourcing, faces challenges like low melting temperatures and mechanical fragility due to its distinct composition. In this study, the focus was on exploring strategies to enhance the stability of fish collagen for potential human applications. To address this, our research investigated a non-covalent binding strategy, by using heat shock protein 47 (Hsp47) from zebrafish and human to stabilize fish collagen. The co-application of Hsp47 dramatically improved the rates of folding and markedly increased the denaturation temperature of fish collagen from 31.7 degrees C to 37.7 degrees C, demonstrating promising outcomes for potential in vitro applications. Optical Nanoscopy Electron Microscopy (OpNS-EM) imaging demonstrates the complex formation between Hsp47 and collagen fibers, shedding light on their interaction dynamics, while cytotoxicity assays affirm the safety of the Collagen-Hsp47 complex on human keratinocytes. This pioneering methodology applies a viable approach to stabilize fish collagen, expanding its potential applications across human-related domains.

Cholestatic liver injury (CLI) is a clinical syndrome caused by impaired bile excretion and is almost asymptomatic in early-stage. If CLI is not recognized timely, it will elicit more severe liver diseases. Therefore, earlier diagnosis of CLI is urgently needed but remains challenging. On the basis of the consensus that elevation of serum alkaline phosphatase (ALP) is the most susceptible indicator of earlier cholestatic diseases. Herein, we conjugated dicyanoisophorone with hydroxyl-substituted coumarin derivative to afford an intramolecular charge transfer (ICT)-based fluorophore TX-OH, and successfully constructed an enzyme activatable fluorescent probe TX-PS, which enables specifically detecting ALP with near-infrared emission (720nm), large stokes shift (198nm) and excellent linear relationship (R2 = 0.9969) between fluorescence intensity and the concentrations of ALP (0-100U/L). Moreover, TX-PS exhibits applicable for imaging of ALP activities in living cells and zebrafish with ignorable toxicity and shows great potential to visually screen ALP inhibitors. Importantly, the liver tissues from CLI mice displayed remarkably higher fluorescence intensity than that from normal mice, suggesting the valuable capability of TX-PS for evaluating CLI degree. Overall, this work affords a powerful tool for earlier diagnosis of CLI and high-throughput screening of ALP inhibitors or new drugs for CLI therapeutics.

Artificial photoenzymes with novel catalytic modes not found in nature are in high demand; yet, they also present significant challenges in the field of biocatalysis. In this study, a chemogenetic modification strategy is developed to facilitate the rapid diversification of photoenzymes. This strategy integrates site-specific chemical conjugation of various artificial photosensitizers into natural protein cavities and the iterative mutagenesis in cell lysates. Through rounds of directed evolution, prominent visible-light-activatable photoenzyme variants were developed, featuring a thioxanthone chromophore. They successfully enabled the enantioselective [2 + 2] photocycloaddition of 2-carboxamide indoles, a class of UV-sensitive substrates that are traditionally challenging for known photoenzymes. Furthermore, the versatility of this photoenzyme is demonstrated in enantioselective whole-cell photobiocatalysis, enabling the efficient synthesis of enantioenriched cyclobutane-fused indoline tetracycles. These findings significantly expand the photophysical properties of artificial photoenzymes, a critical factor in enhancing their potential for harnessing excited-state reactivity in stereoselective transformations.

Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI’s exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.