Enzyme Catalysis Research Articles

Activation and Reactivity of the Deubiquitinylase OTU Cezanne-2 from MD Simulations and QM/MM Calculations.

Cezanne-2 (Cez2) is a deubiquitinylating (DUB) enzyme involved in the regulation of ubiquitin-driven cellular signaling and selectively targets Lys11-linked polyubiquitin chains. As a representative member of the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic isopeptide bond cleavage; however, its exact catalytic mechanism is not yet resolved. In this work, we used different computational approaches to get molecular insights into the Cezanne-2 catalytic mechanism. Extensive molecular dynamics (MD) simulations were performed for 12 μs to model free Cez2 and the diubiquitin (diUb) substrate-bound protein-protein complex in two different charge states of Cez2, each corresponding to a distinct reactive state in its catalytic cycle. The simulations were analyzed in terms of the relevant structural parameters for productive enzymatic catalysis. Reactive diUb-Cez2 complex configurations were identified, which lead to isopeptide bond cleavage and stabilization of the tetrahedral oxyanion intermediate. The reliability of these complexes was further assessed by quantum mechanics/molecular mechanics (QM/MM) optimizations. The results show that Cez2 follows a modified cysteine protease mechanism involving a catalytic Cys210/His367 dyad, with the oxyanion hole to be a part of the "C-loop," and polarization of His367 by the formation of a strictly conserved water bridge with Glu173. The third residue has a dual role in catalysis as it mediates substrate binding and polarization of the catalytic dyad. A similar mechanism was identified for Cezanne-1, the paralogue of Cez2. In general, our simulations provide valuable molecular information that may help in the rational design of selective inhibitors of Cez2 and closely related enzymes.

Combining Photochemical Oxyfunctionalization and Enzymatic Catalysis for the Synthesis of Chiral Pyrrolidines and Azepanes.

Chiral heterocyclic alcohols and amines are frequently used building blocks in the synthesis of fine chemicals and pharmaceuticals. Herein, we report a one-pot photoenzymatic synthesis route for N-Boc-3-amino/hydroxy-pyrrolidine and N-Boc-4-amino/hydroxy-azepane with up to 90% conversions and >99% enantiomeric excess. The transformation combines a photochemical oxyfunctionalization favored for distal C-H positions with a stereoselective enzymatic transamination or carbonyl reduction step. Our study demonstrates a mild and operationally simple asymmetric synthesis workflow from easily available starting materials.

Enzymatic electrosynthesis system based on multi-enzyme catalysis or coupled with microbial transformation

Enzymatic electrosynthesis harnesses electrical energy to drive the production of value-added chemicals through enzymatic catalysis. Although single-enzyme-catalyzed electrosynthesis is commonly used, it often presents challenges for complex chemical syntheses in...

In Silico Strategies for Characterizing Inner Cavities of Lipid-Binding Proteins.

Cavities in proteins perform diverse functions such as substrate binding, enzyme catalysis, passage for transportation of small molecules, and protein oligomerization. Often, the physical properties of these cavities are closely linked to the protein function; such as the hydrophobic lipid-binding cavities in lipid-binding proteins (LBPs) that protect lipid substrates from the larger aqueous milieu. Therefore, the characterization of protein cavities can provide valuable insights into protein structure-function relationships, hinting toward their mechanism of action while aiding in the identification of ligand binding sites that are essential for drug discovery approaches. Several algorithms have historically been designed to identify and characterize the different types of cavities in protein structures. We summarize these algorithms and provide a step-by-step guide for locating and characterizing internal cavities in proteins using CICLOP by using ATP-binding cassette transporter A1 (ABCA1) as an example.

Probe on the pivotal role of green rust in improving the enzymatic activity and facilitating the formation of 1O2 in Fenton-like process for degradation of 4-chlorophenol

Iron-based materials have demonstrated significant efficacy in catalyzing hydrogen peroxide (H2O2) for the removal of antibiotics from aquatic environments. Green rust (GR), a hybrid valence state iron-based catalyst, was synthesized. By exploiting the catalytic properties of glucose oxidase (GOx) to generate H2O2 from glucose (Glu), a GR-GOx/Glu system for the removal of recalcitrant organic compound 4-chlorophenol (4-CP) was constructed. In terms of pollutant degradation efficiency, an increase of 30% was observed compared to Fe2+/H2O2 system. Utilizing density functional theory (DFT), we calculated the electrostatic potential energy and charge density distribution, demonstrating the existence of active electron transfer between GR and flavin adenine dinucleotide (FAD), which subsequently enhanced the activity of GOx. The enhancement was pivotal for the sustained generation of preferred oxidative species and rapid degradation of pollutants within the system. Furthermore, the acids and H2O2 generated during enzyme catalysis not only neutralized the alkalinity released by the Fenton-like reaction but also promoted the conversion of superoxide radical (·O2-) to singlet oxygen (1O2). Overall, this study elucidates the fundamental motivation underlying the enhancement of enzymatic activity and highlights the critical role of 1O2 within the system, providing valuable insights into the potential mechanisms by which metal hydroxides catalyze the H2O2 process.

A quinoline-malononitrile-based fluorescent probe with aggregation-induced emission effect for the monitoring of viscosity in vivo†

As an essential microenvironmental parameter, viscosity controls to some extent the diffusion of molecular species in cells during processes such as signaling, enzyme catalysis and biomolecular interactions. However, abnormal viscosity...

Sensitivity-enhanced self-powered biosensing platform for detection of sugarcane smut using Mn-doped ZIF-67, RCA-DNA nano-grid array and capacitor.

Sugarcane smut is a widespread fungal disease, which severely impairs the quality and sugar yield of sugarcane. Early detection is crucial for mitigating its impact, which makes the development of a highly sensitive and accurate detection method essential. Herein, the Mn-doped zeolite imidazolate framework (ZIF-67), synthesized via a nano-confined-reactor approach, is designed to significantly enhance electron transport and boost the enzyme loading capacity within biofuel cells, thereby potentially enhancing their overall performance. By integrating rolling circle amplification (RCA) with DNA nano-grid array, an sensitive detection platform is engineered based on biofuel cells that can specifically identify the sugarcane smut gene fragment (bE4'). Upon recognition of the target gene bE4', the arm strands of the locked bridge DNA is triggered to open to initiate a series of reactions. This process not only anchores DNA to the electrode, but also promotes RCA under the catalysis of enzymes to produce long single-stranded DNA to capture DNA nano-grid array. The formation of double-stranded DNA can capture [Ru(NH3)6]3+ to further amplify the output signal. Furthermore, a capacitor is integrated into the detection system and a 16.7-fold increase in sensitivity is therefore obtained. In the concentration from 10-4 to 104 pM, the method shows a robust linear response with a detection limit of 34.5 aM (S/N=3). This work presents a dependable, high-sensitivity and portable detection solution for the early and efficient detection of sugarcane smut, exhibiting great potential for agricultural disease management and monitoring.

Mechanisms for translating chiral enantiomers separation research into macroscopic visualization.

Chirality is a common phenomenon in nature, including the dominance preference of small biomolecules, the special spatial conformation of biomolecules, and the biological and physiological processes triggered by chirality. The selective chiral recognition of molecules in nature from up-bottom or bottom-up is of great significance for living organisms. Such as the transcription of DNA, the recognition of membrane proteins, and the catalysis of enzymes all involve chiral recognition processes. The selective recognition between these macromolecules is mainly achieved through non covalent interactions such as hydrophobic interactions, ammonia bonding, electrostatic interactions, metal coordination, van der Waals forces, and π-π stacking. Researchers have been committed to studying how to convert this weak non covalent interaction into macroscopic visualization, which has further understood of the interactions between chiral molecules and is of great significance for simulating the interactions between molecules in living organisms. This article reviews several models of chiral recognition mechanisms, the interaction forces involved in the chiral recognition process, and the research progress of chiral recognition mechanisms. The outlook in this review points out that studying chiral recognition interactions provides an important bridge between chiral materials and the life sciences, providing an ideal platform for studying chiral phenomena in biological systems.

Macrocyclic catalysis mediated by water: opportunities and challenges.

Nanospaces within enzymes play a crucial role in chemical reactions in biological systems, garnering significant attention from supramolecular chemists. Inspired by the highly efficient catalysis of enzymes, artificial supramolecular hosts have been developed and widely employed in various reactions, paving the way for innovative and selective catalytic processes and offering new insights into enzymatic catalytic mechanisms. In supramolecular macrocycle systems, weak non-covalent interactions are exploited to enhance substrate solubility, increase local concentration, and stabilize the transition state, ultimately accelerating reaction rates and improving product selectivity. In this review, we will focus on the opportunities and challenges associated with the catalysis of chemical reactions by supramolecular macrocycles in the aqueous phase. Key issues to be discussed include limitations in molecular interaction efficiency in aqueous media, product inhibition, and the incompatibility of catalysts or conditions in "one-pot" reactions.

Production of 2-O-α-d-glucopyranosyl-l-ascorbic acid using sucrose phosphorylase by semi-rational design

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is often substituted for l-ascorbic acid (L-AA) in health- and skincare products due to its enhanced stability and comparable antioxidant. Enzymatic catalysis for AA-2G is gaining widespread interest and sucrose phosphorylases (SPase) have shown promise in achieving higher yields. To enhance AA-2G synthesis, we screened and identified the SPase from Bifidobacterium longum (BlSPase) as the starting enzyme, with an optimal pH of 5.4 and temperature of 45 °C for AA-2G synthesis. Subsequently, we conducted semi-rational design on BlSPase based on modeling and molecular docking. L-AA was docked with the BlSPase model to analyze key residues in the ‘loop-door’ domain and substrate-binding pocket. Through alanine scanning and saturation mutagenesis, a mutant library was created. After single-point and composite mutation, L341V/V346P was identified as the optimal variant. Ultimately, within 72 h, we achieved yield of 358.6 g/L AA-2G with a molar conversion of L-AA reaching 75.7 %.

Bis-pyrrolidone structures as versatile building blocks for the synthesis of bio-based polyesters and for the preparation of additives.

In this work, three bis-pyrrolidone-based structures (BP) were synthesized combining dimethyl itaconate (DMI), the dimethyl ester derivative of itaconic acid, with various aliphatic diamines having a C4 to C12 carbon chain length with the aim of developing novel bio-based building blocks. All three BPs were obtained with a purity >93% and could further be used without performing any tedious purification step, therefore allowing an easy scalability of the synthesis on a 10 g scale. Their potential application was demonstrated in two key areas of modern polymer science: (1) the enzymatic synthesis of polyesters and (2) their use as poly(lactic acid) (PLA) additives. Firstly, the possibility of obtaining oligoesters by reacting the BP monomers with various aliphatic diols in a solventless reaction system and under mild conditions (T < 90 °C) was demonstrated thanks to the use of enzymatic catalysis. Linear oligoesters having mean average molecular weights between 1000 g mol-1 and 6100 g mol-1 and dispersity values <2 were successfully obtained. When applying the BP structures as PLA additives, the incorporation of a 10% w w-1 BP in the polyester matrix resulted in systems with an 8× increased elongation at break and a decrease in the glass transition temperature compared to the neat polymer matrix.

The Bifunctional Enzymatic Catalysis of Maize Waste for Simultaneous Production of Arabinose and Xylose

The Bifunctional Enzymatic Catalysis of Maize Waste for Simultaneous Production of Arabinose and Xylose

Chemoenzymatic synthesis of Tamsulosin.

Several chemoenzymatic pathways have been developed for the stereoselective production of the drug tamsulosin. The interest in the exclusive synthesis of its (R)-enantiomer lies in the greater activity compared to that displayed by its (S)-counterpart for the treatment of kidney stones and benign prostatic hyperplasia disease. Using different types of biocatalysts such as lipases, alcohol dehydrogenases and transaminases, three complementary strategies have been studied to introduce chirality into a key synthetic precursor. The first approach involved the lipase-catalyzed kinetic resolution of a racemic amine precursor, although low conversions and selectivities were found. A second strategy consisted in the synthesis of a chiral alcohol intermediate through a bioreduction proccess catalyzed by ADHs, with the identification of stereocomplementary redox enzymes capable of producing both enantiomers. The (S)-alcohol, obtained with ADH-A from Rhodococcus ruber, was subsequently converted into the corresponding amine through a telescoped approach. Alternatively, transaminases were also employed for the biotransamination of the previously studied intermediate ketone, which led directly to the enantiopure (R)-amine in high yield. Finally, the active pharmaceutical ingredient was prepared in enantiopure form and in 49% overall yield from the ketone precursor by a two-step sequential transformation of the chiral amine building block. These findings highlight the importance and versatility of enzyme catalysis for the stereoselective synthesis of drugs.

Electrochemical kinetic fingerprinting of single-molecule coordinations in confined nanopores.

Metal centers are essential for enzyme catalysis, stabilizing the active site, facilitating electron transfer, and maintaining the structure through coordination with amino acids. In this study, K238H-AeL nanopores with histidine sites were designed as single-molecule reactors for the measurement of single-molecule coordination reactions. The coordination mechanism of Au(III) with histidine and glutamate in biological nanopore confined space was explored. Specifically, Au(III) interacts with the nitrogen (N) atom in the histidine imidazole ring of the K238H-AeL nanopore and the oxygen (O) atom in glutamate to form a stable K238H-Au-Cl2 complex. The formation mechanism of this complex was further validated through single-molecule nanopore analysis, mass spectrometry, and molecular dynamics simulations. Introducing histidine and negative charge amino acids with carboxyl group into different positions within the nanopore revealed that the formation of the histidine-Au coordination bond in the confined space requires a suitable distance between the ligand and the central metal atom. By analyzing the association and dissociation rates of the single Au(III) ion under the applied voltages, it was found that a confined nanopore increased the bonding rate constant of Au(III)-histidine coordination reactions by around 10-100 times compared to that in the bulk solution and the optimal voltage for single-molecule. Therefore, nanopore techniques for tracking single-molecule reactions could offer valuable insights into designing metalloenzymes in metal-catalyzed organic reactions.

Recent advances in the organocatalytic synthesis of chiral C3-spiro-cyclopentaneoxindoles.

Although there have been various reviews on the asymmetric construction of C3-spirooxindoles, there is a scarcity of reviews focusing on the asymmetric organocatalytic synthesis of C3-spiro-cyclopentaneoxindole derivatives. This particular scaffold has garnered significant attention from synthetic chemists due to its relevance in medicinal chemistry. In this review, we provide an overview of recent advancements in the asymmetric organocatalytic synthesis of various C3-spiro-cyclopentaneoxindoles using organic catalysts. The work is divided into sections according to the type of catalysis, including covalent catalysis (amine catalysis and N-heterocyclic carbene catalysis) and non-covalent catalysis (chiral phosphoric acid catalysis, hydrogen bond catalysis, and phase transfer catalysis). Furthermore, we discuss existing challenges and future directions within this field. It is our belief that this review will serve as an informative resource for researchers engaged in synthesizing C3-spiro-cyclopentaneoxindoles and inspire further advancements in this area.

SUCLG1 promotes aerobic respiration and progression in plexiform neurofibroma.

Plexiform neurofibromas (PNFs) are benign tumors that affect 20‑50% of patients with type I neurofibromatosis (NF1). PNF carries a risk of malignancy. There is no effective cure for PNF. Its onset may be associated with genetic and metabolic abnormalities, but the exact mechanisms remain unclear. Succinate‑CoA ligase GDP/ADP‑Forming Subunit α(SUCLG1), a catalytic enzyme in the tricarboxylic acid cycle, is highly expressed in PNF. The present study aimed to explore the role of SUCLG1 in function and metabolism of PNF cells. SUCLG1 expression was verified using western blotting and immunofluorescence. After inducing SUCLG1 knockdown and overexpression, functional changes in PNF cells were assessed, as well as effects of SUCLG1 on cell respiration and glucose metabolism. Quantitative PCR, WB, electron microscopy and Flow cytometry demonstrated that SUCLG1 enhanced mitochondrial quality and promoted mitochondrial fusion, thereby driving proliferation and migration of tumor cells, inhibiting apoptosis and altering the cell cycle. A Seahorse assay showed that elevated SUCLG1 expression enhanced cell aerobic respiration without affecting the glycolytic process. This suggests that SUCLG1 upregulation in PNF does not trigger the Warburg effect associated with malignant tumors. This study also demonstrated the positive regulation of cellular function by promoting the expression level of the SLC25A1 gene when SUCLG1 expression was elevated. In conclusion, SUCLG1 altered the mechanism of mitochondrial quality control to enhance cell aerobic respiration, thereby driving the pathogenesis of PNF. Thus, SUCLG1 can serve as a potential target in future therapeutic strategies.

Anomalous Role of Carbon in Pd‐Catalyzed Selective Hydrogenation

AbstractCarbonaceous species, including subsurface carbidic carbon and surface carbon, play crucial roles in heterogeneous catalysis. Many reports suggested the importance of subsurface carbon in the selective hydrogenation of alkynes over Pd‐based catalysts. However, the role of surface carbon has been largely overlooked. We demonstrate that subsurface carbon in Pd is not responsible for the selectivity in acetylene hydrogenation. In contrast, the structure of surface carbonaceous species plays a decisive role in hydrogenation selectivity. Electron microscopy and spectroscopy evidence, along with theoretical modelling, reveal that partial graphitization of surface carbonaceous species results in unique spatial confinement of surface reaction intermediates, thus altering the reaction energy landscape in favour of ethylene desorption as opposed to over‐hydrogenation. This mechanism for selectivity control is analogous to enzyme catalysis, where the active centers selectively attract reactants and release products. Similar mechanism may be present in CO/CO2 hydrogenation and alkane dehydrogenation reactions.

Hyperglycemia-responsive nitric oxide-releasing biohybrid cryogels with cascade enzyme catalysis for enhanced healing of infected diabetic wounds.

Diabetic wound infections are a frequent complication for diabetic patients, and conventional treatment for combating diabetic wound infections relies on antibiotics. However, the misuse and overuse of antibiotics have led to the emergence of drug-resistant bacteria, making these infections challenging to treat. Thus, there is an urgent need for alternative strategies to effectively manage diabetic wound infections. Herein, we have developed a hyperglycemia-responsive antibacterial cryogel system that can generate and release hydrogen peroxide (H2O2) and nitric oxide (NO). This system involves incorporating glucose oxidase (GO) and L-Arginine (L-Arg: A) into hyaluronic acid aldehyde methacryloyl (HAAMA: H) and gelatin methacryloyl (GelMA: G) hybrid cryogels (GOA@HG). HAAMA facilitated higher loading and longer stability of L-Arg and GO via a Schiff base reaction. In vitro studies demonstrate that GOA@HG cryogels exhibited outstanding breathability, effective exudate management, and excellent hemostasis capabilities. Moreover, this system could consume excess glucose in diabetic wounds and efficiently eliminate bacteria through the cascaded release of H2O2 and NO without causing antibiotic resistance. In vivo studies further reveal that GOA@HG cryogels significantly enhanced the healing of infected diabetic wounds by inhibiting bacterial growth, accelerating blood vessel formation, and promoting collagen deposition. Overall, GOA@HG cryogels displayed remarkable wound dressing properties and synergistic antimicrobial effects owing to glucose-responsive H2O2 and NO release, which could serve as a highly efficient therapeutic strategy for treating infected diabetic wounds.

Entrapment of Lipase from Candida antarctica in a Xerogel for the Production of Biodiesel from Waste Cooking Oil.

The process to synthesize biodiesel is well-developed and optimized to overcome the disadvantages like the competition with agriculture using feedstock, and the problematics in the process. Oils from waste and enzymatic catalysis have proven to be good solutions to these problems. Lipases are currently the most commonly used enzymes in the transesterification of oils; nevertheless, enzymes have a high cost and must be immobilized to offer repetitive reuse. This work focuses on the synthesis and the optimization of the conditions of a xerogel where the lipase from Candida antarctica is immobilized by entrapment. The xerogel synthesized here, a glass-like material, proved to be very effective in maintaining the enzyme on its structure, with an immobilization yield of 96 % and an activity of 5.3 U/mg (free lipase=12.1 U/mg). By grinding this xerogel, the immobilization yield remained constant (94 %). Nevertheless, grinding the xerogel allowed better diffusion of reagents, and the activity reached 9.7 U/mg. Using this xerogel in biodiesel production had a positive effect on CALB efficiency by increasing the biodiesel yield, 46.1 % against 15.1 % with free CALB. Finally, the catalyst could be reused for five runs without loss of activity.

MMLV reverse transcriptase, a relevant tool for molecular biology

The reverse transcriptase of Moloney Murine Leukemia Virus (MMLV) is an enzyme that synthesizes DNA from an RNA template. Among reverse transcriptases, this enzyme is currently the most commonly used in molecular biology and diagnostics. Since its discovery, this viral protein has been extensively studied, shedding light on its structural and functional characteristics, and offering opportunities to optimize the catalytic performances for biotechnological applications. The review describes the structural motifs of the reverse transcriptase of MMLV, the key amino acids in enzymatic catalysis and the enzyme's properties such as processivity, fidelity and thermal stability. This review reports the optimizations made to improve these parameters, which enabled the modified enzymes to be integrated in the molecular biology tests used in the laboratory on a daily basis.