ACS Chemical Biology最新文献

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from Marasmius rotula (MroUPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of MroUPO, a UPO gene from Marasmius wettsteinii (MweUPO-1), that has a similar reaction profile to MroUPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant MweUPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.

Nickel-based catalysts demonstrate promising potential in integrated hydrogen production via methanol electro-oxidation (MOR). However, the MOR involves multiple hydroxide ions (OH^–) and multielectron synergistic catalytic processes in alkaline electrolytes. The low OH^– capture capability of Ni-based catalysts leads to a reduced energy conversion efficiency. Furthermore, the competitive adsorption of H₂O and CH₃OH molecules on the catalyst surface blocks active sites, resulting in a decreased selectivity for formate. To address these challenges, effectively manipulating the electrochemical microenvironment has emerged as a viable strategy. In this study, we successfully achieved selective electrooxidation of methanol to formate on Ni(OH)₂ by incorporating a hard Lewis acid heteroatom (Cr) to finely tune the electrochemical interface microenvironment. Experimental and theoretical investigations reveal that incorporating ordered Cr atoms into Ni(OH)₂ can establish a hydrophobic interface, suppressing the blockage of active sites and promoting the enrichment of OH^– at the electrified interface. By leveraging the enhanced localized alkalinity and hydrophobic microenvironment at the modified electrified interface, high-value formate can be effectively synthesized with nearly 100% selectivity over a wide potential range. Furthermore, the catalysts display robust electrocatalytic capabilities, delivering remarkable performance with a high current density of 50 mA cm^–2 at a working potential of 1.45 V vs RHE.

Recent advancements in AI-driven computational modeling, especially AlphaFold2, have revolutionized the prediction of biological macromolecule structures. AlphaFold2 enabled accurate predictions of structural domains and complex arrangements. However, computational models lack a clear metric for accuracy. This study explores whether computational models can match the crystallographic resolution of crystal structures. By comparing distances between atoms in models and crystal structures using t tests, it was found that AlphaFold2 models are comparable to high-resolution crystal structures (1.1 to 1.5 Å). While these models exhibit exceptional quality, their accuracy is lower than the crystal structure with resolutions better than 1 Å.

Protein misfolding and aggregation are the hallmarks of neurodegenerative diseases including Huntington's disease, Parkinson's disease, Alzheimer's disease, and prion diseases. A crowded cellular environment plays a crucial role in modulating protein aggregation processes in vivo and the pathological aggregation of proteins linked to different neurodegenerative disorders. Here, we review recent studies examining the effects of various crowding agents, such as polysaccharides, polyethylene glycol, and proteins like BSA and lysozyme on the behaviors of aggregation of several amyloidogenic peptides and proteins, including amylin, huntingtin, tau, α-synuclein, prion, and amyloid-β. We also summarize how the aggregation kinetics, thermodynamic stability, and morphology of amyloid fibrils are altered significantly in the presence of crowding agents. In addition, we also discuss the molecular basis underlying the modulation of amyloidogenic aggregation, focusing on changes in the protein conformation, and the nucleation mechanism. The molecular understanding of the effects of macromolecular crowding on amyloid aggregation is essential for revealing disease pathologies and identifying possible therapeutic targets. Thus, this review offers a perspective on the complex interplay between protein aggregation and the crowded cellular environment in vivo and explains the relevance of crowding in the context of neurodegenerative disorders.

Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca²⁺ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% ΔF/F per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% ΔF/F per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca²⁺-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca²⁺ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca²⁺ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.

Electrochemical CO₂ reduction (CO₂R) allows us to close the carbon cycle and store intermittent renewable energy into chemical products. Among these, syngas, a mixture of hydrogen and carbon monoxide, is particularly valuable due to its high market share and the low energy required for its electrocatalytic production. In addition to catalyst optimization, lately, electrolyte modifications to achieve a suitable CO/H₂ ratio have also been considered. Ionic liquid (IL)-based electrolytes have enabled high faradaic efficiency toward CO, depending on the chemical properties of the IL. In this work, we rationalized through density functional theory (DFT) descriptors the competition between hydrogen evolution (HER) and CO₂R on silver in imidazolium-based electrolytes, developing a DFT-based analytical model. The electrolyte anion regulates the concentration ratio between cationic and carbene species of ILs cation, respectively, between the 1-ethyl-3-methylimidazolium cation (EMIM⁺) and carbene (EMIM:) species and between the 1-butyl-3-methylimidazolium cation (BMIM⁺) and carbene (BMIM:). The latter species, if formed, hinders the CO₂R by blocking the active sites or trapping CO₂ in solution. In the case of weak Lewis base anions as fluorinated ones, EMIM⁺ (BMIM⁺) cations, which serve as cocatalysts in CO₂R, are more abundant, allowing high CO partial current densities and high electrochemically active surface area. Applying the here-defined descriptors to ILs not yet tested makes it possible to predict the HER and CO₂R selectivity on silver, thus enabling guidelines for designing better ILs for CO₂R.

While enzymatic epoxidation of activated olefins by P450s has been well-established, chemo- and enantioselective epoxidation of unactivated olefins remains a formidable challenge, mainly due to the presence of competing hydroxylation of allylic C–H bonds. In addition, P450 monooxygenase-catalyzed epoxidation of olefins generally provides S-configured products with high enantiopurity, and examples of P450 enzymes demonstrating high R-enantioselectivity in epoxidation reactions remain rare. Herein, we report a chemoselective and enantiodivergent epoxidation of unactivated alkenes using engineered P450DA monooxygenases. The P450DA variants, obtained through structure-guided directed evolution based on the X-ray of P450DA-WT and P450DA-M3, switch the reactivity from the native hydroxylation of the allylic C–H bonds to epoxidation of C═C bonds and exhibit superior chemoselectivity (up to 99% epoxidation selectivity) and enantioselectivity (up to >99:1 er), delivering a wide variety of versatile and enantioenriched epoxides. Notably, an enantiodivergent synthesis was achieved simply by employing different P450DA variants, leading to both enantiomers of the epoxide products. Various transformations of the products were carried out, illustrating the synthetic utilities of the methods. Furthermore, molecular dockings and molecular dynamics simulations reveal the origin of high epoxidation selectivity and complementary stereoselectivity of the mutants.

The synthetic potential of unsymmetrically substituted arynes is not yet fully realized due to regioselectivity issues. Although many models exist to predict the regioselectivity of arynes, these models do not hold for metal-mediated reactions. Previously, we reported a way to induce regioselectivity in a metal-catalyzed aryne annulation reaction by using bulky monodentate phosphine ligands. Reported herein is a mechanistic investigation into the operative catalytic cycle within this transformation. Additionally, the molecular parameters responsible for regioselectivity have been examined via linear free energy relationships and multivariate linear regression. This model shows the interdependence on both the steric and electronic properties of the aryne and the size of the phosphine ligand for regioselectivity.

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule “correctors” for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10–20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.