Enzymatic Reaction Research Articles

Efficient Enzymatic Glycan Engineering of Extracellular Vesicles Using Nanomaterial-Interfaced Microfluidics.

Extracellular vesicles (EVs) present a promising modality for numerous biological and medical applications, including therapeutics. Developing facile methods to engineer EVs is essential to meeting the rapidly expanding demand for various functionalized EVs in these applications. Herein, we developed a technology that integrates enzymatic glycoengineering and microfluidics for effective EV functionalization. This method builds on a 3D nanostructured microfluidic device to streamline a multiple-step EV engineering process, which involves a step of enzymatic reaction to install azido-sialic acid residues to glycans on EVs using a sialyltransferase and an azide-tagged sialyl donor followed by the attachment of various functionalities, such as biotin and fluorescent labels, to the resulting azido-glycans on EVs through a biocompatible click reaction. Compared to traditional EV engineering methods, we show that our technology improves the efficiency of EV glycoengineering while simplifying and expediting the workflow. Furthermore, we demonstrated the applicability of this technology to EVs derived from the cell lines of different cancer types, including A549, PC3, and COLO-1 cells. Overall, this EV engineering technology could provide a potentially useful tool for broad applications.

Extraction of E171 or E172 food additives from sugar-based or lipid-based complex matrices for primary particles identification by electron microscopy

Measurement of primary particle size distributions is fundamental to determining if a material should be classified as a nanomaterial according to the European Commission recommended definition. This is of particular relevance to alimentary products, where current regulation requires that any additives containing nanoforms be explicitly declared as such in the ingredients’ list. Currently, Electron Microscopy is the only instrumental technique able to reliably classify materials as nanomaterials. To verify the nanostatus of a material using this technique, it is essential to firstly extract the additives from the matrix in which they are dispersed, and then measure a wide size distribution while avoiding artefacts from the food matrix. In this operating procedure, extraction protocols, developed and applied for sugar-based or lipid-based food, are described. In particular, case studies for titanium dioxide E171 and iron oxide/iron hydroxide based materials such as E172 are considered. The protocols are based on gentle extraction of the additive materials and could, in principle, be used for other nanomaterials provided they are not soluble in water or ethanol and are resistant to enzymatic reactions. The size distribution of the extracted material can then be determined from images acquired using scanning and/or transmission electron microscopy.

Gain of pancreatic beta cell-specific SCD1 improves glucose homeostasis by maintaining functional beta cell mass under metabolic stress.

The key pancreatic beta cell transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA) is critical for the maintenance of mature beta cell function and phenotype. The expression levels and/or activities of MafA are reduced when beta cells are chronically exposed to diabetogenic stress, such as hyperglycaemia (i.e. glucotoxicity). Interventional targets and adjuvant therapies to abate MafA loss in beta cells may provide evidence to support the effective treatment of diabetes. In this study, we aimed to investigate the function of stearoyl-CoA desaturase 1 (SCD1) in the stabilisation of MafA expression and activity in order to maintain functional beta cell mass, with a view to suppressing the development of type 2 diabetes. SCD1 expression levels were analysed in islets obtained from humans with type 2 diabetes, hyperglycaemic db/db mice, and a high-fat diet (HFD)-induced mouse model of diabetes. Pancreatic beta cell-specific Scd1 knockin (βSCD1KI) mice were generated to study the role of SCD1 in beta cell function and identity. The protein-to-protein interactions between SCD1 and MafA were detected in MIN6 and HEK293A cells. We used experiments including chromatin immunoprecipitation, cell-based ubiquitination assay and fatty acid composition analysis to investigate the specific molecular mechanism underlying the effect of SCD1 on the restoration of MafA and beta cell function under glucotoxic conditions. SCD1 expression was reduced in beta cells of humans with type 2 diabetes and in HFD-fed and db/db mice compared with healthy controls, which was attributed to glucotoxicity-induced Scd1 promoter histone deacetylation. Gain-of-function of SCD1 in beta cells improved insulin deficiency, glucose intolerance and beta cell dedifferentiation/transdifferentiation in the HFD-induced mouse model of diabetes. Mechanistically, SCD1 directly bound to the E3 ubiquitin ligase HMG-CoA reductase degradation 1 (HRD1) and stabilised nuclear MafA through interrupting MafA-HRD1 interactions in mouse islets and MIN6 cells, which inhibited the ubiquitination-mediated degradation of MafA. Moreover, the products of SCD enzyme reactions (mainly oleic acid) also alleviated glucotoxicity-mediated oxidative stress in MIN6 cells. Our findings indicate that SCD1 stabilises beta cell MafA both in desaturase-dependent and -independent manners, thus improving glucose homeostasis under metabolic stress. This provides a potential novel target for precision medicine for the treatment of diabetes.

Water Migration through Enzyme Tunnels Is Sensitive to the Choice of Explicit Water Model.

The utilization of tunnels and water transport within enzymes is crucial for their catalytic function as water molecules can stabilize bound substrates and help with unbinding processes of products and inhibitors. Since the choice of water models for molecular dynamics simulations was shown to determine the accuracy of various calculated properties of the bulk solvent and solvated proteins, we have investigated if and to what extent water transport through the enzyme tunnels depends on the selection of the water model. Here, we focused on simulating enzymes with various well-defined tunnel geometries. In a systematic investigation using haloalkane dehalogenase as a model system, we focused on the well-established TIP3P, OPC, and TIP4P-Ew water models to explore their impact on the use of tunnels for water molecule transport. The TIP3P water model showed significantly faster migration, resulting in the transport of approximately 2.5 times more water molecules compared to that of the OPC and 1.7 times greater than that of the TIP4P-Ew. Finally, the transport was 1.4-fold more pronounced in TIP4P-Ew than in OPC. The increase in migration of TIP3P water molecules was mainly due to faster transit times through dehalogenase tunnels. We observed similar behavior in two different enzymes with buried active sites and different tunnel network topologies, i.e., alditol oxidase and cytochrome P450, indicating that our findings are likely not restricted to a particular enzyme family. Overall, this study showcases the critical importance of water models in comprehending the use of enzyme tunnels for small molecule transport. Given the significant role of water availability in various stages of the catalytic cycle and the solvation of substrates, products, and drugs, choosing an appropriate water model may be crucial for accurate simulations of complex enzymatic reactions, rational enzyme design, and predicting drug residence times.

Enzymatic Reaction Network-driven Polymerization-induced Transient Coacervation.

A living cell has a highly complex microenvironment whereas numerous enzyme-driven processes are active at once. These procedures are incredibly accurate and efficient, although comparable control has not yet been established in vitro. Here, we design an enzymatic reaction network (ERN) that combines antagonistic and orthogonal enzymatic networks to produce adjustable dynamics of ATP-fueled transient coacervation. Using horseradish peroxidase (HRP)-mediated Biocatalytic Atom Transfer Radical Polymerization (BioATRP), we synthesized poly(dimethylaminoethyl methacrylate), which subsequently formed coacervates with ATP. We rationally explored enzymatic control over coacervation and dissolution, using orthogonal and antagonistic enzyme pairs viz., alkaline phosphatase, Creatine phosphokinase, hexokinase, esterase, and urease. ATP-fuelled coacervates also demonstrate the enzymatic catalysis to prove its potential to be exploited as a cellular microreactor. Additionally, we developed ERN-polymerization-induced transient coacervation (ERN-PIC), with complete control over the system, polymerization, coacervation, and dissolution. Notably, the coacervation process itself determines functional properties, as seen in selective cargo uptake. The strategy offers cutting-edge biomimetic applications, and insights into cellular compartmentalization by bridging the gap between synthetic and biological systems. The development of temporally programmed coacervation is promising for the spatial arrangement of multienzyme cascades, and offers novel ideas on the architecture of artificial cells.

Parahydrogen Polarization in Reverse Micelles and Application to Sensing of Protein-Ligand Binding.

A medium containing reverse micelles supports non-hydrogenative parahydrogen induced polarization (nhPHIP) in the organic phase while solubilizing a protein in the aqueous phase. Strongly enhanced NMR signals from iridium hydride complexes report on a ligand, 4-amino-2-benzylaminopyrimidine, which crosses the phase boundary and interacts with the thiaminase protein TenA. The calculation of binding equilibria reveals a KD of 39.7 ± 8.9 μM for protein binding. The nanoscale separation of the two phases allows the separate optimization of the parahydrogen polarization and solubilization of a biological macromolecule. The reverse micelles may be used to study other biological questions using signal enhancement by parahydrogen polarization, such as enzyme reactions, protein-protein interactions, and protein binding epitopes.

Chemoenzymatic Synthesis Planning Guided by Reaction Type Score.

Thanks to the growing interest in computer-aided synthesis planning (CASP), a wide variety of retrosynthesis and retrobiosynthesis tools have been developed in the past decades. However, synthesis planning tools for multistep chemoenzymatic reactions are still rare despite the widespread use of enzymatic reactions in chemical synthesis. Herein, we report a reaction type score (RTscore)-guided chemoenzymatic synthesis planning (RTS-CESP) strategy. Briefly, the RTscore is trained using a text-based convolutional neural network (TextCNN) to distinguish synthesis reactions from decomposition reactions and evaluate synthesis efficiency. Once multiple chemical synthesis routes are generated by a retrosynthesis tool for a target molecule, RTscore is used to rank them and find the step(s) that can be replaced by enzymatic reactions to improve synthesis efficiency. As proof of concept, RTS-CESP was applied to 10 molecules with known chemoenzymatic synthesis routes in the literature and was able to predict all of them with six being the top-ranked routes. Moreover, RTS-CESP was employed for 1000 molecules in the boutique database and was able to predict the chemoenzymatic synthesis routes for 554 molecules, outperforming ASKCOS, a state-of-the-art chemoenzymatic synthesis planning tool. Finally, RTS-CESP was used to design a new chemoenzymatic synthesis route for the FDA-approved drug Alclofenac, which was shorter than the literature-reported route and has been experimentally validated.

Discovery and Functional Identification of 2,3-Oxidosqualene Cyclases and Cytochrome P450s in Triterpenoid Metabolic Pathways of Actinidia eriantha.

Actinidia eriantha Benth, known as the "king of fruits", is rich in triterpenoid compounds, particularly ursane-type and oleanane-type triterpenic acids. These secondary metabolites have been widely applied in medicine, cosmetics, agriculture, and other fields. To date, key enzyme genes involved in triterpenoid metabolic pathways in A. eriantha remain unexplored. This study employed transcriptome sequencing analysis combined with synthetic biology approaches involving heterologous expression in yeast to identify crucial genes responsible for the biosynthesis of triterpenoid components in A. eriantha: Two 2,3-oxidosqualene cyclases (AeOSC2 and AeOSC3) were characterized to catalyze the formation of major triterpene scaffolds, α-amyrin [precursor of ursolic acid (UA)], β-amyrin [precursor of oleanolic acid (OA)], and ψ-taraxasterol, and two cytochrome P450s (AeCYP716A8 and AeCYP716A9) mediating three-step oxidation at the C-28 position of ursane-type and oleanane-type triterpene scaffolds to form UA, OA, and intermediate oxidation products. We successfully reconstructed the biosynthetic pathway of ursane- and oleanane-type triterpenoids from A. eriantha in a heterologous yeast host and elucidated the two-step enzymatic reactions involved in triterpenoid biosynthesis. These findings lay the foundation for further understanding the biosynthesis of key active components in A. eriantha.

Photothermal Nanozyme‐Encapsulating Microneedles for Synergistic Treatment of Infected Wounds

AbstractPhotothermal therapy (PTT) is widely used in wound healing because it produces heat under near‐infrared irradiation, eliminating bacterial infections. However, excessive production of reactive oxygen species (ROS) occurs during PTT, which causes oxidative stress damage. In this study, a nanozyme, PDA‐MnO2 (PM), is synthesized and encapsulated in the needle tip layer of a bi‐layer GelMA microneedle patch (PbMP), which has excellent mechanical properties and can easily penetrate the skin. In a study involving an SD rat wound model infected with S. aureus, the PbMP significantly accelerated the healing process of bacterially challenged wounds, resulting in better blood vessel growth, cell proliferation, and collagen biosynthesis than other wound dressings. The experiment shows that the PM nanoparticles in PbMP improve photothermal conversion efficiency and activate SOD and CAT, resulting in an efficient cascade enzyme reaction system to eliminate ROS during the PTT process. This study describes a highly effective nanozyme hydrogel microneedle platform with photothermal synergistic antibacterial effects on wound healing.

Ultrasmall High-Entropy-Alloy Nanozyme Catalyzed In Vivo ROS and NO Scavenging for Anti-Inflammatory Therapy.

High-entropy alloy (HEA) nanoparticles possess finely tunable and multifunctional catalytic activity due to their extremely diverse adsorption sites. Their unique properties enable HEA nanoparticles to mimic the complex interactions of the redox homeostasis system, which is composed of cascade and multiple enzymatic reactions. The application of HEAs in mimicking complex enzymatic systems remains relatively unexplored, despite the importance of regulating biological redox reactions. Here, it is reported that ultra-small (<10 nm in a diameter) HEA nanozymes consisting of five platinum-group metals with tunable morphologies from planar to dendritic structures are synthesized. The synthesized HEA nanozymes exhibited higher peroxidase-like activity compared to monometallic platinum-group nanoparticles. Additionally, HEA nanoparticles effectively mimicked RONS-regulation metabolism in cascade reactions involving superoxide dismutase and catalase, as well as in multiple reactions including HORAC and NO scavenging. As a result, the HEA nanozyme exhibited superior anti-inflammatory efficacy both in vitro and in vivo. The findings underscore the effectiveness of the high-entropy alloy structure in restoring in vivo enzymatic systems through intrinsic activity enhancements and cascade reaction mechanisms.

Simultaneous Observation of the Anomerization and Reaction Rates of Enzymatic Dehydrogenation of Glucose-6-Phosphate by Dissolution DNP.

The hyperpolarization of biological samples using dissolution dynamic nuclear polarization (dDNP) has become an attractive method for the monitoring of fast chemical and enzymatic reactions using NMR by taking advantage of a large signal increase. This approach is actively developing but still needs key methodological breakthroughs to be used as an analytical method for the monitoring of complex networks of simultaneous metabolic pathways. In this article, we use the deceptively simple example of glucose-6-phosphate (G6P) oxidation reaction by the enzyme G6P dehydrogenase (G6PDH) to discuss some important methodological aspects of dDNP kinetic experiments, such as its robustness and its ability to provide repeatable results as well as the capacity of this time-resolved methodology to test kinetic models and hypotheses and to provide reliable parameter estimates. To illustrate the potential of our approach, we report the first direct and quantitative evidence of selectivity of G6PDH toward the β anomer of G6P.

Ultrasensitive SERS Profiling of Intracellular Hydrogen Peroxide Release Based on Enzymatic Amplification and Silent-Region Raman Reporter.

Surface-enhanced Raman scattering spectroscopy (SERS) has been widely applied to screen biomarkers and biological species due to its high sensitivity; however, severe background noise signals significantly limit its practical application. In this study, we develop an ultrasensitive SERS sensor to determine cellular oxidative stress based on the H2O2-induced enzymatic amplification and a silent-range Raman fingerprint. In the presence of horseradish peroxidase, H2O2 could trigger the coupling reaction of 4-hydroxythiophenol (4-MTP) with phenol-d5 to form a new compound, which can bind to the SERS substrate via the Au-S bond and generate the stable SERS signal with nearly zero background signals owing to the Raman-silent fingerprint of phenol-d5 at 2125 cm-1. The high-performance enzymatic reaction and the silent-range Raman fingerprint enable the ultrasensitive determination of H2O2 with a broad linear range of 5 × 10-9 to 1 × 10-3 M and a limit of detection as low as 1.6 nM. Taking advantage of its excellent sensitivity and anti-interference capability, the as-developed SERS sensor is employed to profile the triggered dynamic change of the intracellular H2O2 release, and further to testify the cancerous cells exhibiting a significantly higher level of intracellular oxidative stress than the healthy cells, and the different cell populations possessing the susceptible difference to additional oxidative stimulation. This study paves the way for improving SERS sensing capability through the silent-range fingerprint and signal amplification via an enzymatic reaction and reveals SERS as an effective tool for monitoring the endogenous oxidative behavior of living cells.

Engineering a Silk Protein-Mediated Customizable Compartment for Modular Metabolic Synthesis.

Microbial cell factories provide a nontoxic, economical way for the synthesis of various chemicals and drugs, garnering significant attention from researchers. However, excessive dispersion of enzymes and accumulation of intermediate metabolites in the production process will weaken the reaction efficiency of the pathway enzyme. In this study, a cellular compartment was constructed to isolate the enzyme reaction space and optimize the modular metabolic synthesis. First, a special spider silk protein was designed and constructed to form protein condensates in microbial cells, and its synthetic microcompartment effects were investigated. Second, the interaction of short peptide pairs or direct fusion based on the silk protein was used to recruit a variety of enzymes to improve the efficiency of enzyme catalysis. Third, the 2'-fucosyllactose (2'-FL) de novo synthesis pathway and its modular optimization were carried out to verify the mode. Finally, a synthetic compartment was introduced into the pathway to directly aggregate the 2'-FL synthesis pathway, thus obtaining synthetic-compartment-mediated multienzyme aggregates. The experimental results showed that the titer of 2'-FL was significantly improved compared with those of wild-type and modular-optimized free enzymes. The utilization of this cell microcompartment offers a novel avenue for the aggregation of diverse enzymes, thereby offering an innovative approach for enhancing the efficiency of the microbial modular metabolic pathway.

Efficient and Cost-Effective Synthesis of N-Acetyllactosamine by Sequential Modular Enzymatic Cascade Reactions Involving NTP Regeneration.

Human milk oligosaccharides (HMOs) play important roles in the development of infants, which are the third most abundant component in human milk. N-Acetyllactosamine (LacNAc) is an important intermediate for the biosynthesis of other HMOs and antigens. Since currently appropriate synthetic methods for large-scale production of LacNAc are not available, it is urgently needed to develop an efficient and cost-effective synthetic pathway for LacNAc preparation. In this study, a cost-effective pathway of LacNAc synthesis involving regeneration of adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP) was established. After optimizing the reaction conditions, LacNAc was synthesized at a yield of >90% via a sequential one-pot multienzyme (OPME) method with crude enzymes at 100 mM substrates. Finally, LacNAc was produced efficiently and cost-effectively at a 5 L scale. This strategy would possibly meet the requirements of potential industrial production of LacNAc and would provide guidance for the production of other structurally complex HMOs or functional oligosaccharides in the future.

Solvent-free synthesis of citronellyl acetate using Fermase: Optimization, kinetics and RSM studies

Citronellyl acetate is a flavor ester with a fresh fruit aroma and a fruity, sweet flavor. The current study illustrates the synthesis of citronellyl acetate via a transesterification reaction of citronellol and vinyl acetate in a solvent-free system with the help of biocatalyst Fermase. The effect of various parameters affecting the reaction, including reactant mole ratio, enzyme loading, temperature, and agitation speed, was studied to obtain the maximum conversion of citronellol. Under optimized conditions with a 1:10 mole ratio of citronellol to vinyl acetate, 3% (w/w) enzyme loading with respect to citronellol, 300 RPM agitation speed, and 60 °C temperature, a maximum conversion of 99.8% was achieved in 2 h reaction time. The enzyme was reused for six reaction cycles, retaining 85% of its activity. Various kinetic models for enzymatic reactions were explored and validated. The ping-pong bi-bi model with both substrate inhibition gave the best fit for the experimental data. Optimization studies were also conducted using the response surface methodology to observe the interaction of various parameters.

Boosting Enzyme-like Activities via Atomization of Cerium for Tumor Microenvironment-Responsive Cascade Therapy.

Nanozyme catalytic therapeutic efficacy is limited by the finite enzyme activity and specificity. In this work, nitrogen-doped carbon loaded with a cerium single-atom nanozyme (Ce SAs@NC) is synthesized, exhibiting tumor specificity and excellent multiple enzyme-like activities. Compared with nitrogen-doped carbon loaded with CeO2 nanoparticles, Ce SAs@NC shows excellent peroxidase-like and catalase-like activity. Ce SAs@NC can convert intracellular hydrogen peroxide into cytotoxic hydroxyl radical and O2, which can be further transferred to superoxide radicals. Cascade enzyme reactions not only alleviate the hypoxic microenvironment of tumors but also induce lipid peroxidation and apoptosis or necrosis of tumor cells. The mild photothermal action will enhance the enzyme-like activities of Ce SAs@NC rather than induce the production of heat shock proteins to protect tumor cells. In addition, Ce SAs@NC can regulate the immune environment, stimulate M1 macrophages to trigger immune responses, and inhibit tumor proliferation. Thanks to the combination of the size effect of the single atoms, photothermal influence, multiple enzyme-like activities, and immunological effect, the Ce SAs@NC platform appears to have tumor specificity, less toxic side effects, and a high curative effect both in vitro and in vivo.

Transglutaminase-triggered dual gradients of mechanical and biochemical cues self-assembling peptide hydrogel for guiding MC3T3-E1 cell behaviors

The mechanical properties and bioactive motif densities of extracellular matrix materials play crucial roles in regulating cell behaviors, such as cell adhesion, migration, proliferation, and differentiation. However, current studies on cellular responses to ECM predominantly concentrated on polymer hydrogels featuring a single factor, such as the mechanical strength, the types of bioactive motifs, and the morphology of the polymers. This limited focus may overlook the complex interplay of multiple factors. Here, we developed dual gradient peptide Q3GT-I3K hydrogels with tunable mechanical strength (0.3–4.0 kPa) and different density of bioactive motif (0.45–3.67 mM) by enzymatic crosslinking. These hydrogels can mimic the viscoelasticity of natural soft tissues. The properties of mechanical strength and cell responsive motif density could be controlled by modulating the proportion of the substrates in the enzymatic reaction. MC3T3 cells significantly differentiated into osteoblasts after seeded on the Q3GT-I3K hydrogel (2.8 kPa, 1.83 mM Q3GT) for 21 days, identifying from the elevated expression of alkaline phosphatase and substantial calcium nodule formation. Importantly, the engineered hydrogels exert a synergistic effect on the cell behaviors such as early adhesion, late proliferation, and differentiation of MC3T3-E1 cells. This paper introduces a new strategy for designing tissue engineering scaffold materials with specific functions.

CeO2/NH2-MIL-88B(Fe) composites with peroxidase-like activity for colorimetric detection and photo-enzymatic synergetic degradation of ciprofloxacin hydrochloride

Photocatalysis combined with enzyme catalysis for wastewater treatment is now a promising and challenging process. Therefore, it is necessary to synthesize a material with both photocatalytic and enzyme-catalyzed functions. CeO2/NH2-MIL-88B(Fe) composites with peroxidase-like activity (referred to as CNF-X, where X is the molar ratio of Ce to Fe) were prepared using a solvothermal method. The composites can be used for colorimetric detection, the limit of detection (LOD) was 0.029µM in the range of 50-200µM of ciprofloxacin hydrochloride (CIP). Using a combination of photocatalytic and enzymatic reactions, light excites enzyme activity to achieve highly efficient and selective chemical reactions. In addition, H2O2 activation promotes redox cycling reactions between Ce and Fe, providing more active sites and facilitating photo-enzymatic synergetic catalysis. Under the conditions of 0.01g CNF-5, pH = 3.5, and 25 °C, more than 92.8% of CIP (20mg/L solution) was degraded within 80min, and the photo-enzyme synergistic catalysis mechanism was examined. The stable enzymatic activity and good reusability of the material were confirmed by cycling experiments and X-ray diffraction (XRD) patterns before and after the reaction. The contribution ratio of radicals and non-radicals in the degradation of CIP was analyzed. Density functional theory (DFT) calculation was applied to predict possible degradation pathways. Simulation results stemmed from Toxicity Estimation Software Tool (T.E.S.T) showed an overall reduction in the toxicity of the intermediates. This study provides important application value and theoretical guidance for peroxidase-like materials in environmental detection and remediation.

Cryo-EM Detection of AMPylated Histidine Implies Covalent Catalysis in AMPylation Mediated by a Bacterial Effector.

AMPylation is a post-translational modification (PTM) whereby adenosine monophosphate (AMP) from adenosine triphosphate (ATP) is transferred onto protein hydroxyl groups of serine, threonine, or tyrosine. Recently, an actin-dependent AMPylase namely LnaB from the bacterial pathogen Legionella pneumophila was found to AMPylate phosphate groups of phosphoribosylated ubiquitin and Src family kinases. LnaB represents an evolutionarily distinct family of AMPylases with conserved active site Ser-His-Glu residues. Here, we capture the structure of the LnaB-actin complex in a putative intermediate state via single-particle cryogenic electron microscopy (cryo-EM) and find that the catalytic histidine of LnaB is covalently attached to AMP through a phosphoramidate linkage at the Nδ1 atom. This observation provides direct structural evidence of histidine AMPylation as a PTM and implies the possibility of covalent catalysis in LnaB-mediated AMPylation, a mechanism distinct from known AMPylases. Subsequent biochemical studies confirm the observed AMP binding site and provide additional insights into the catalytic properties of LnaB. Together, our work highlights the power of cryo-EM in capturing labile PTMs and transient species during enzymatic reactions, while opening new avenues of mechanistic investigation into the LnaB family.

Glucuronidation of orally administered drugs and the value of nanocarriers in strategies for its overcome.

The gastrointestinal tract (GIT) plays a pivotal role in the absorption of orally administered drugs, with the small intestine serving as the primary site due to its extensive surface area and specialized cell types, including enterocytes and M cells. After oral administration, drugs are generally transported via the portal vein to the liver, where they undergo first-pass metabolism. This process involves various enzymatic reactions, including glucuronidation, facilitated by uridine diphosphate-glucuronosyltransferase (UGT), a major phase 2 reaction in mammalian metabolism. UGTs conjugate glucuronic acid to a wide array of endogenous and exogenous substrates, enhancing their solubility and excretion, but significantly affecting the bioavailability and therapeutic efficacy of drugs. UGT enzymes are ubiquitously distributed across tissues, prominently in the liver, but also in the GIT, kidneys, brain, and other organs where they play crucial roles in xenobiotic metabolism. Species-specific differences in UGT expression and activity impact the selection of animal models for pharmacological studies. Various experimental models - ranging from computational simulations (in silico) to laboratory experiments (in vitro) and animal studies (in vivo) - are employed throughout drug discovery and development to evaluate drug metabolism, including UGT activity. Effective strategies to counter pre-systemic metabolism are critical for improving drug bioavailability. This review explores several approaches including prodrugs, co-administration of specific molecules or use of inhibiting excipients in formulations. Strategies incorporating these excipients in nanoformulations demonstrate notable increases in drug absorption and bioavailability. This review highlights the importance of targeted delivery systems and excipient selection in overcoming metabolic barriers, aiming to optimize drug efficacy and patient outcomes.