RESUMEN
ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.
Asunto(s)
ADP Ribosa Transferasas , Proteínas Bacterianas , Modelos Moleculares , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , ADP-Ribosilación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chromobacterium/química , Chromobacterium/enzimología , Chromobacterium/genética , Cristalografía por Rayos X , NAD/química , NAD/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
Cellular redox homeostasis is essential for maintaining cellular activities, such as DNA synthesis and gene expression. Inspired by this, new therapeutic interventions have been rapidly developed to modulate the intracellular redox state using artificial transmembrane electron transport. However, current approaches that rely on external electric field polarization can disrupt cellular functions, limiting their in vivo application. Therefore, it is crucial to develop novel electric-field-free modulation methods. In this work, we for the first time found that graphene could spontaneously insert into living cell membranes and serve as an electron tunnel to regulate intracellular reactive oxygen species and NADH based on the spontaneous bipolar electrochemical reaction mechanism. This work provides a wireless and electric-field-free approach to regulating cellular redox states directly and offers possibilities for biological applications such as cell process intervention and treatment for neurodegenerative diseases.
Asunto(s)
Membrana Celular , Grafito , Oxidación-Reducción , Especies Reactivas de Oxígeno , Grafito/química , Humanos , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/química , Transporte de Electrón , Membrana Celular/metabolismo , Membrana Celular/química , NAD/química , NAD/metabolismo , ElectronesRESUMEN
The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by â¼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.
Asunto(s)
Dihidroorotato Deshidrogenasa , Lactococcus lactis , Biocatálisis , Catálisis , Dominio Catalítico , Mononucleótido de Flavina/metabolismo , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/metabolismo , Flavina-Adenina Dinucleótido/química , Cinética , Lactococcus lactis/enzimología , Lactococcus lactis/metabolismo , NAD/metabolismo , NAD/química , Oxidación-ReducciónRESUMEN
NAD(H)-dependent enzymes play a crucial role in the biosynthesis of pharmaceuticals and fine chemicals, but the limited recyclability of the NAD(H) cofactor hinders its more general application. Here, we report the generation of mechano-responsive PEI-modified Cry3Aa protein crystals and their use for NADH recycling over multiple reaction cycles. For demonstration of its practical utility, a complementary Cry3Aa protein particle containing genetically encoded and co-immobilized formate dehydrogenase for NADH regeneration and leucine dehydrogenase for catalyzing the NADH-dependent l-tert-leucine (l-tert-Leu) biosynthesis has been produced. When combined with the PEI-modified Cry3Aa crystal, the resultant reaction system could be used for the efficient biosynthesis of l-tert-Leu for up to 21 days with a 10.5-fold improvement in the NADH turnover number.
Asunto(s)
Formiato Deshidrogenasas , NAD , NAD/metabolismo , NAD/química , Formiato Deshidrogenasas/metabolismo , Formiato Deshidrogenasas/química , Leucina-Deshidrogenasa/metabolismo , Leucina-Deshidrogenasa/química , Cristalización , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Modelos MolecularesRESUMEN
Artificial photosynthesis represents a sustainable strategy for accessing high-value chemicals; however, the conversion efficiency is significantly limited by its difficulty in the cycle of coenzymes such as NADH. In this study, we report a series of isostructural triazine covalent organic frameworks (COFs) and explore their N-substituted microenvironment-dependent photocatalytic activity for NADH regeneration. We discovered that the rational alteration of N-heterocyclic species, which are linked to the triazine center through an imine linkage, can significantly regulate both the electron band structure and planarity of a COF layer. This results in different separation efficiencies of the photoinduced electron-hole pairs and electron transfer behavior within and between individual layers. The optimal COF catalyst herein achieves an NADH regeneration capacity of 89% within 20 min, outperforming most of the reported nanomaterial photocatalysts. Based on this, an artificial photosynthesis system is constructed for the green synthesis of a high-value compound, L-glutamate, and its conversion efficiency significantly surpasses the enzymatic approach without the NADH photocatalytic cycle. This work offers new insights into the coenzyme regeneration by means of regulating the distal heterocyclic microenvironment of a COF skeleton, holding great potential for the green photosynthesis of important chemicals.
Asunto(s)
Estructuras Metalorgánicas , Triazinas , Triazinas/química , Catálisis , Estructuras Metalorgánicas/química , NAD/química , NAD/metabolismo , Procesos Fotoquímicos , Estructura Molecular , Coenzimas/química , Coenzimas/metabolismo , FotosíntesisRESUMEN
Human-borne acetone is a potent marker of lipid metabolism. Here, an enzyme immobilization method for secondary alcohol dehydrogenase (S-ADH), which is suitable for highly sensitive and selective biosensing of acetone, was developed, and then its applicability was demonstrated for spatiotemporal imaging of concentration distribution. After various investigations, S-ADH-immobilized meshes could be prepared with less than 5% variation by cross-linking S-ADH with glutaraldehyde on a cotton mesh at 40 °C for 15 min. Furthermore, high activity was obtained by adjusting the concentration of the coenzyme nicotinamide adenine dinucleotide (NADH) solution added to the S-ADH-immobilized mesh to 500 µM and the solvent to a potassium phosphate buffer solution at pH 6.5. The gas imaging system using the S-ADH-immobilized mesh was able to image the decrease in NADH fluorescence (ex 340 nm, fl 490 nm) caused by the catalytic reaction of S-ADH and the acetone distribution in the concentration range of 0.1-10 ppm-v, including the breath concentration of healthy people at rest. The exhaled breath of two healthy subjects at 6 h of fasting was quantified as 377 and 673 ppb-v, which were consistent with the values quantified by gas chromatography-mass spectrometry.
Asunto(s)
Acetona , Pruebas Respiratorias , Enzimas Inmovilizadas , Acetona/análisis , Acetona/química , Humanos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Técnicas Biosensibles , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Gases/química , Gases/análisis , Espiración , NAD/análisis , NAD/química , NAD/metabolismoRESUMEN
Dihydro-nicotinamide adenine dinucleotide (NADH) detection is crucial since it is a vital coenzyme in organism metabolism. Compared to the traditional method based on natural NADH oxidase (NOX), nanozymes with multienzyme-like activity can catalyze multistage reactions in a singular setup, simplifying detection processes and enhancing sensitivity. In this study, an innovative NADH detection method was developed using iron-doped carbon (Fe@C) nanozyme synthesized from metal-organic frameworks with in situ reduced Pt clusters. This nanozyme composite (Pt/Fe@C) demonstrated dual NOX and peroxidase-like characteristics, significantly enhancing the catalytic efficiency and enabling NADH conversion to NAD+ and H2O2 with subsequent detection. The collaborative research involving both experimental and theoretical simulations has uncovered the catalytic process and the cooperative effect of Fe and Pt atoms, leading to enhanced oxygen adsorption and activation, as well as a decrease in the energy barrier of the key step in the H2O2 decomposition process. These findings indicate that the catalytic performance of Pt/Fe@C in NOX-like and POD-like reactions can be significantly improved. The colorimetric sensor detects NADH with a limit of detection as low as 0.4 nM, signifying a breakthrough in enzyme-mimicking nanozyme technology for precise NADH measurement.
Asunto(s)
Carbono , Estructuras Metalorgánicas , NAD , Platino (Metal) , NAD/química , Estructuras Metalorgánicas/química , Platino (Metal)/química , Carbono/química , Hierro/química , Peróxido de Hidrógeno/química , Colorimetría/métodos , Humanos , Catálisis , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Materiales Biomiméticos/química , Límite de Detección , NADH NADPH OxidorreductasasRESUMEN
NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 µg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.
Asunto(s)
Colorimetría , NAD(P)H Deshidrogenasa (Quinona) , NADH NADPH Oxidorreductasas , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/química , Humanos , NADH NADPH Oxidorreductasas/metabolismo , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/química , Cobalto/química , Carbono/química , Biomimética , Límite de Detección , Nitrógeno/química , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Ferrocianuros/química , NAD/metabolismo , NAD/químicaRESUMEN
Nanozyme-based metabolic regulation triggered by tumor-specific endogenous stimuli has emerged as a promising therapeutic strategy for tumors. The current efficacy, however, is constrained by the limited concentration of endogenous substrates and the metabolic plasticity of tumors. Consequently, the implementation of efficient metabolic regulation in tumor therapy is urgently needed. Herein, a versatile nanozyme-based nicotinamide adenine dinucleotide (NADH) circulating oxidation nanoreactor is reported. First, the synthesized cobalt-doped hollow carbon spheres (Co-HCS) possess NADH oxidase (NOX)-mimicking activity for the NADH oxidation to disrupt oxidative phosphorylation (OXPHOS) pathway of tumor cells. Second, the substrate-cycle manner of Co-HCS can be used for NADH circulating oxidation to overcome the limitation of substrate deficiency. Finally, 2-Deoxy-D-glucose (2-DG) and 6-aminonicotinamide (6-AN) are introduced to block glycolysis and pentose phosphate pathway (PPP), thus creating a versatile nanozyme-based NADH circulating oxidation nanoreactor (Co-HCS/D/A) for tumor therapy through triple cellular metabolism disruption. In vitro and in vivo results demonstrate that the designed nanoreactor not only enhances the catalytic efficiency but also disrupts the tumor metabolic homeostasis, leading to efficient therapy outcome. This study develops a novel NADH circulating oxidation nanoreactor for tumor therapy through triple cellular metabolism disruption, which addresses the limitations of current nanozyme-based metabolism regulation for tumor therapy.
Asunto(s)
NAD , Oxidación-Reducción , NAD/metabolismo , NAD/química , Humanos , Animales , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Línea Celular Tumoral , RatonesRESUMEN
Cellular senescence, a vulnerable state of growth arrest, has been regarded as a potential strategy to weaken the resistance of tumor cells, leading to dramatic improvements in treatment efficacy. However, a selective and efficient strategy for inducing local tumor cellular senescence has not yet been reported. Herein, piezoelectric catalysis is utilized to reduce intracellular NAD+ to NADH for local tumor cell senescence for the first time. In detail, a biocompatible nanomedicine (BTO/Rh-D@M) is constructed by wrapping the piezoelectric BaTiO3/(Cp*RhCl2)2 (BTO/Rh) and doxorubicin (DOX) in the homologous cytomembrane with tumor target. After tumors are stimulated by ultrasound, negative and positive charges are generated on the BTO/Rh by piezoelectric catalysis, which reduce the intracellular NAD+ to NADH for cellular senescence and oxidize H2O to reactive oxygen species (ROS) for mitochondrial damage. Thus, the therapeutic efficacy of tumor immunogenic cell death-induced chemo-immunotherapy is boosted by combining cellular senescence, DOX, and ROS. The results indicate that 23.9% of the piezoelectric catalysis-treated tumor cells senesced, and solid tumors in mice disappeared completely after therapy. Collectively, this study highlights a novel strategy to realize cellular senescence utilizing piezoelectric catalysis and the significance of inducing tumor cellular senescence to improve therapeutic efficacy.
Asunto(s)
Senescencia Celular , Doxorrubicina , Inmunoterapia , Especies Reactivas de Oxígeno , Senescencia Celular/efectos de los fármacos , Animales , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Catálisis , Especies Reactivas de Oxígeno/metabolismo , Ratones , Inmunoterapia/métodos , NAD/metabolismo , NAD/química , Línea Celular Tumoral , Humanos , Titanio/química , Titanio/farmacología , Neoplasias/terapia , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Rodio/química , Rodio/farmacología , Compuestos de BarioRESUMEN
Ferritins are multimeric nanocage proteins that sequester/concentrate excess of free iron and catalytically synthesize a hydrated ferric oxyhydroxide bio-mineral. Besides functioning as the primary intracellular iron storehouses, these supramolecular assemblies also oversee the controlled release of iron to meet physiologic demands. By virtue of the reducing nature of the cytosol, reductive dissolution of ferritin-iron bio-mineral by physiologic reducing agents might be a probable pathway operating in vivo. Herein, to explore this reductive iron-release pathway, a series of quinone analogs differing in size, position/nature of substituents and redox potentials were employed to relay electrons from physiologic reducing agent, NADH, to the ferritin core. Quinones are well known natural electron/proton mediators capable of facilitating both 1/2 electron transfer processes and have been implicated in iron/nutrient acquisition in plants and energy transduction. Our findings on the structure-reactivity of quinone mediators highlight that iron release from ferritin is dictated by electron-relay capability (dependent on E1/2 values) of quinones, their molecular structure (i.e., the presence of iron-chelation sites and the propensity for H-bonding) and the type/amount of reactive oxygen species (ROS) they generate in situ. Juglone/Plumbagin released maximum iron due to their intermediate E1/2 values, presence of iron chelation sites, the ability to inhibit in situ generation of H2O2 and form intramolecular H-bonding (possibly promotes semiquinone formation). This study may strengthen our understanding of the ferritin-iron-release process and their significance in bioenergetics/O2-based cellular metabolism/toxicity while providing insights on microbial/plant iron acquisition and the dynamic host-pathogen interactions.
Asunto(s)
Ferritinas , Hierro , NAD , Oxidación-Reducción , Quinonas , Especies Reactivas de Oxígeno , Ferritinas/química , Ferritinas/metabolismo , Hierro/metabolismo , Hierro/química , NAD/metabolismo , NAD/química , Oxígeno/metabolismo , Oxígeno/química , Quinonas/química , Quinonas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , MycobacteriumRESUMEN
Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.
Asunto(s)
Magnesio , NAD , Niacinamida , Oxidación-Reducción , NAD/química , NAD/metabolismo , Magnesio/química , Niacinamida/química , Niacinamida/análogos & derivados , Sitios de Unión , Cinética , Mononucleótido de Nicotinamida/química , Mononucleótido de Nicotinamida/metabolismo , Compuestos de Piridinio/química , Azul de Metileno/química , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Transporte de ElectrónRESUMEN
Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of ß-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.
Asunto(s)
NAD , NAD/química , Biocatálisis , Catálisis , Cinética , AdsorciónRESUMEN
Reduced nicotinamide adenine dinucleotide (NADH)-detecting electrochemical sensors are attractive in monitoring and diagnosing various physiological disorders of NADH abnormalities. The NADH detection methods using conventional electrodes are challenging due to slow electron transfer and fouling effect. Interestingly, paper-based flexible and disposable electrodes (PE) are superior for sensing biomolecules through simple detection procedures with excellent sensitivity and selectivity. Herein, to construct a conducting polypeptide-modified paper electrode, initially, polytyrosine (PTyr) is synthesized from l-tyrosine N-carboxy anhydride through ring-opening polymerization, and PTyr is drop-coated on the PE. The PTyr-modified paper electrode (PMPE) demonstrated excellent electrochemical properties and facilitated the electrooxidation of NADH at a lower potential of 576 mV. The PMPE displayed a linear detection between 25 and 145 µM of NADH concentration, with a lower detection limit of 0.340 µM. Under ideal circumstances, the sensor developed displayed an excellent NADH detection capability without interference with the most common electroactive species, ascorbic acid. The PMPE facilitates good electrocatalytic activity toward NADH, which can also be employed as a substrate material for biofuel cells.
Asunto(s)
Electrodos , NAD , Papel , NAD/análisis , NAD/química , Péptidos/química , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Oxidación-Reducción , Límite de Detección , Técnicas Biosensibles/métodosRESUMEN
This work presents the synthesis and characterization of an innovative F,S-doped carbon dots/CuONPs hybrid nanostructure obtained by a direct mixture between F,S-doped carbon dots obtained electrochemically and copper nitrate alcoholic solution. The hybrid nanostructures synthesized were characterized by absorption spectroscopy in the Ultraviolet region (UV-vis), high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and different electrochemical techniques. The fluoride and sulfur-doped carbon dots/CuONPs nanostructures were used to prepare a non-enzymatic biosensor on a printed carbon electrode, exhibiting excellent electrocatalytic activity for the simultaneous determination of NADH, dopamine, and uric acid in the presence of ascorbic acid with a detection limit of 20, 80, and 400 nmol L-1, respectively. The non-enzymatic biosensors were also used to determine NADH, dopamine, and uric acid in plasma, and they did not suffer significant interference from each other.
Asunto(s)
Técnicas Biosensibles , Carbono , Cobre , Dopamina , Técnicas Electroquímicas , Límite de Detección , NAD , Ácido Úrico , Ácido Úrico/sangre , Ácido Úrico/química , Técnicas Biosensibles/métodos , Dopamina/sangre , Dopamina/análisis , Carbono/química , NAD/química , NAD/sangre , Cobre/química , Técnicas Electroquímicas/métodos , Humanos , Azufre/química , Fluoruros/química , Puntos Cuánticos/química , Nanoestructuras/química , ElectrodosRESUMEN
A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.
Asunto(s)
Biocatálisis , Oxidación-Reducción , Procesos Fotoquímicos , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Estructura Molecular , NAD/química , NAD/metabolismo , Biomimética , Niacinamida/química , Niacinamida/metabolismo , Compuestos de Nitrógeno/química , GrafitoRESUMEN
In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway catalyzing the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-pyrophosphate (PRPP) to produce nicotinamide mononucleotide (NMN). Given the pivotal role of NAD+ in a range of cellular functions, including DNA synthesis, redox reactions, cytokine generation, metabolism, and aging, NAMPT has become a promising target for many diseases, notably cancer. Therefore, various NAMPT inhibitors have been reported and classified as first and second-generation based on their chemical structures and design strategies, dual-targeted being one. However, most NAMPT inhibitors suffer from several limitations, such as dose-dependent toxicity and poor pharmacokinetic properties. Consequently, there is no clinically approved NAMPT inhibitor. Hence, research on discovering more effective and less toxic dual-targeted NAMPT inhibitors with desirable pharmacokinetic properties has drawn attention recently. This review summarizes the previously reported dual-targeted NAMPT inhibitors, focusing on their design strategies and advantages over the single-targeted therapies.
Asunto(s)
Antineoplásicos , Inhibidores Enzimáticos , Neoplasias , Nicotinamida Fosforribosiltransferasa , Animales , Humanos , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Estructura Molecular , Neoplasias/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Relación Estructura-Actividad , NAD/química , NAD/metabolismo , Niacinamida/químicaRESUMEN
Here we demonstrate the preparation of enzyme-metal biohybrids of NAD+ reductase with biocatalytically-synthesised small gold nanoparticles (NPs, <10â nm) and core-shell gold-platinum NPs for tandem catalysis. Despite the variety of methods available for NP synthesis, there remains a need for more sustainable strategies which also give precise control over the shape and size of the metal NPs for applications in catalysis, biomedical devices, and electronics. We demonstrate facile biosynthesis of spherical, highly uniform, gold NPs under mild conditions using an isolated enzyme moiety, an NAD+ reductase, to reduce metal salts while oxidising a nicotinamide-containing cofactor. By subsequently introducing platinum salts, we show that core-shell Au@Pt NPs can then be formed. Catalytic function of these enzyme-Au@Pt NP hybrids was demonstrated for H2-driven NADH recycling to support enantioselective ketone reduction by an NADH-dependent alcohol dehydrogenase.
Asunto(s)
Biocatálisis , Oro , Nanopartículas del Metal , NAD , Platino (Metal) , Nanopartículas del Metal/química , NAD/química , NAD/metabolismo , Oro/química , Platino (Metal)/química , Hidrógeno/química , Hidrógeno/metabolismo , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/química , Oxidación-ReducciónRESUMEN
The direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD+) results in various products, complicating the regeneration of the crucial 1,4-NADH cofactor for enzymatic reactions. Previous research primarily focused on steady-state polarization to examine potential impacts on product selectivity. However, this study explores the influence of dynamic conditions on the selectivity of NAD+ reduction products by comparing two dynamic profiles with steady-state conditions. Our findings reveal that the main products, including 1,4-NADH, several dimers, and ADP-ribose, remained consistent across all conditions. A minor by-product, 1,6-NADH, was also identified. The product distribution varied depending on the experimental conditions (steady state vs. dynamic) and the concentration of NAD+, with higher concentrations and overpotentials promoting dimerization. The optimal yield of 1,4-NADH was achieved under steady-state conditions with low overpotential and NAD+ concentrations. While dynamic conditions enhanced the 1,4-NADH yield at shorter reaction times, they also resulted in a significant amount of unidentified products. Furthermore, this study assessed the potential of using pulsed electrochemical regeneration of 1,4-NADH with enoate reductase (XenB) for cyclohexenone reduction.
Asunto(s)
Técnicas Electroquímicas , NAD , Oxidación-Reducción , NAD/química , NAD/metabolismoRESUMEN
Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.