Decreased in Mitochondrial Complex I Subunit NDUFS2 Is Critical for Oocyte Quality During Postovulatory Aging in Pigs.

The levels of nicotinamide adenine dinucleotide (NADH) dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2, a subunit of NADH dehydrogenase) decrease in aged tissues, and these reductions may be partly associated with age-related conditions such as Parkinson's disease. Aging leads to many mitochondrial defects, such as biogenesis disruption, dysfunction, defects in the mitochondrial membrane potential, and production of reactive oxygen species, that may be highly related to NDUFS2 expression. The relationship between NDUFS2 and postovulatory oocyte aging in pigs remains unknown. In this study, we investigated changes in NDUFS2 expression during postovulatory aging (POA). Furthermore, NDUFS2 was knocked down via dsRNA microinjection at the MII stage to evaluate the effects on mitochondrial-related processes during POA. The mRNA expression of NDUFS2 decreased significantly after 48-h aging compared with that in fresh oocytes. NDUFS2 knockdown (KD) significantly impaired the maintenance of oocyte morphology and blastocyst development of embryos after POA. The levels of PGC1α (mitochondrial biogenesis-related proteins) decreased significantly after NDUFS2 KD, while the level of GSNOR, a protein denitrosylase, was reduced by NDUFS2 KD after 48 h of aging. These data suggest that NDUFS2 is vital for maintaining the oocyte quality during POA in pigs.

The compartment-specific manipulation of the NAD+/NADH ratio affects the metabolome and the function of glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

Electro-Driven Multi-Enzymatic Cascade Conversion of CO2 to Ethylene Glycol in Nano-Reactor.

Multi-enzymatic cascade reaction provides a new avenue for C─C coupling directly from CO2 under mild conditions. In this study, a new pathway with four enzymes including formate dehydrogenase (PaFDH), formaldehyde dehydrogenase (BmFADH), glycolaldehyde synthase (PpGALS), and alcohol dehydrogenase (GoADH) is developed for directly converting CO2 gas molecules to ethylene glycol (EG) in vitro. A rhodium-based NADH regeneration electrode is constructed to continuously provide the proton and electron of this multi-enzymatic cascade reaction. The prepared electrode can reach the Faradaic Efficiency (FE) of 82.9% at -0.6 V (vs. Ag/AgCl) and the NADH productivity of 0.737 mM h-1. Shortening the reaction path is crucial for multi-enzymatic cascade reactions. Here, a hydrogen-bonded organic framework (HOF) nano-reactor is successfully developed to immobilize four enzymes in one pot with a striking enzyme loading capacity (990 mg enzyme g-1 material). Through integrating and optimization of NADH electro-regeneration and enzymatic catalysis in one pot, 0.15 mM EG is achieved with an average conversion rate of 7.15 × 10-7 mmol CO2 min-1 mg-1 enzymes in 6 h. These results shed light on electro-driven multi-enzymatic cascade conversion of C─C coupling from CO2 in the nano-reactor.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells.

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

OsLdh7, a rice lactate dehydrogenase, confers stress resilience in rice under cadmium stress through NAD+/NADH regulation.

Lactate dehydrogenase (Ldh, EC 1.1.1.27), an oxidoreductase enzyme catalyses the interconversion of pyruvate to L-lactate and vice-versa with concomitant oxidation and reduction of NADH and NAD+. The enzyme functions as a ROS sensor and mitigates stress response by maintaining NAD+/NADH homeostasis. In this study, we delineated the role of the Ldh enzyme in imparting cadmium stress tolerance in rice. Previously, we identified a putatively active Ldh in rice (OsLdh7) through insilico modelling. Biochemical characterization of the OsLdh7 enzyme revealed it to be optimally active at pH 6.6 in the forward direction and pH 9 in the reverse direction. Overexpression of OsLdh7 in rice cv. IR64, increased tolerance of the transgenic lines to cadmium stress compared to the wild type (WT) at both seedling and reproductive stages. The transgenic lines showed increased enzyme activity in the reverse direction under cadmium stress, attributed to elevated cytosolic pH resulting from increased calcium concentration. This increased NADH content is highly essential for functioning of the ROS scavenging enzymes, RbohD and MPK6. qPCR analysis revealed that the overexpression lines had increased transcript abundance of these genes indicating an effective ROS scavenging mechanism. Additionally, the overexpression lines showed an efficient cadmium sequestration mechanism compared to the WT by increasing the transcript levels of the vacuolar transporters of cadmium as well as total phytochelatin content. Thus, our findings indicated OsLdh7 imparts cadmium stress tolerance in rice through a two-pronged approach by mitigating ROS and sequestering cadmium ions, highlighting its potential for crop improvement programs.

Discovery of antibacterial diketones against gram-positive bacteria.

The rapid rise of antibiotic resistance calls for the discovery of new antibiotics with distinct antibacterial mechanisms. New target mining is indispensable for developing antibiotics. Plant-microbial antibiotics are appealing to underexplored sources due to a dearth of comprehensive understanding of antibacterial activity and the excavation of new targets. Here, a series of phloroglucinol derivatives of plant-root-associated Pseudomonas fluorescens were synthesized for structure-activity relationship analysis. Notably, 2,4-diproylphloroglucinol (DPPG) displayed efficient bactericidal activity against a wide range of gram-positive bacteria. Importantly, mechanistic study exhibits that DPPG binds to type II NADH dehydrogenase (NDH-2), an essential enzyme catalyzing the transfer of electrons from NADH to quinones in the electron transport chain (ETC), blocking electron transfer in S. aureus. Last, we validated the efficacy of DPPG in vivo through animal infection models. Our findings not only provide a distinct antibiotic lead to treat multidrug resistant pathogens but also identify a promising antibacterial target.

Functionalized Metal-organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

NAD metabolism and heart failure: Mechanisms and therapeutic potentials.

Nicotinamide adenine dinucleotide provides the critical redox pair, NAD+ and NADH, for cellular energy metabolism. In addition, NAD+ is the precursor for de novo NADP+ synthesis as well as the co-substrates for CD38, poly(ADP-ribose) polymerase and sirtuins, thus, playing a central role in the regulation of oxidative stress and cell signaling. Declines of the NAD+ level and altered NAD+/NADH redox states have been observed in cardiometabolic diseases of various etiologies. NAD based therapies have emerged as a promising strategy to treat cardiovascular disease. Strategies that reduce NAD+ consumption or promote NAD+ production have repleted intracellular NAD+ or normalized NAD+/NADH redox in preclinical studies. These interventions have shown cardioprotective effects in multiple models suggesting a great promise of the NAD+ elevating therapy. Mechanisms for the benefit of boosting NAD+ level, however, remain incompletely understood. Moreover, despite the robust pre-clinical studies there are still challenges to translate the therapy to clinic. Here, we review the most up to date literature on mechanisms underlying the NAD+ elevating interventions and discuss the progress of human studies. We also aim to provide a better understanding of how NAD metabolism is changed in failing hearts with a particular emphasis on types of strategies employed and methods to target these pathways. Finally, we conclude with a comprehensive assessment of the challenges in developing NAD-based therapies for heart diseases, and to provide a perspective on the future of the targeting strategies.

Disruption of glucose homeostasis by bacterial infection orchestrates host innate immunity through NAD+/NADH balance.

Metabolic reprogramming is crucial for activating innate immunity in macrophages, and the accumulation of immunometabolites is essential for effective defense against infection. The NAD+/NADH (ratio of nicotinamide adenine dinucleotide and its reduced counterpart) redox couple serves as a critical node that integrates metabolic pathways and signaling events, but how this metabolite couple engages macrophage activation remains unclear. Here, we show that the NAD+/NADH ratio serves as a molecular signal that regulates proinflammatory responses and type I interferon (IFN) responses divergently. Salmonella Typhimurium infection leads to a decreased NAD+/NADH ratio by inducing the accumulation of NADH. Further investigation shows that an increased NAD+/NADH ratio correlates with attenuated proinflammatory responses and enhanced type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to intensified proinflammatory responses and restrained type I IFN responses. These results show that the NAD+/NADH ratio is an essential cell-intrinsic factor that orchestrates innate immunity, which enhances our understanding of how metabolites fine-tune innate immunity.

Assessing Breast Cancer through Tumor Microenvironment Mapping of Collagen and Other Biomolecule Spectral Fingerprints─A Review.

Breast cancer is a major challenge in the field of oncology, with around 2.3 million cases and around 670,000 deaths globally based on the GLOBOCAN 2022 data. Despite having advanced technologies, breast cancer remains the major type of cancer among women. This review highlights various collagen signatures and the role of different collagen types in breast tumor development, progression, and metastasis, along with the use of photoacoustic spectroscopy to offer insights into future cancer diagnostic applications without the need for surgery or other invasive techniques. Through mapping of the tumor microenvironment and spotlighting key components and their absorption wavelengths, we emphasize the need for extensive preclinical and clinical investigations.

Correlation between intracellular electron transfer and gene expression for electrically conductive pili in electroactive bacteria during anaerobic digestion with ethanol.

Ethanol feeding has been widely documented as an economical and effective strategy for establishing direct interspecies electron transfer (DIET) during anaerobic digestion. However, the mechanisms involved are still unclear, especially on correlation between intracellular electron transfer in electroactive bacteria and their gene expression for electrically conductive pili (e-pili), the most essential electrical connection component for DIET. Upon cooling from room temperature, the conductivity of digester aggregates with ethanol exponentially increased by an order of magnitude (from 45.5 to 125.4 µS/cm), whereas which with its metabolites (acetaldehyde [from 40.5 to 54.4 µS/cm] or acetate [from 32.1 to 50.4 µS/cm]) did not increase significantly. In addition, the digester aggregates only with ethanol were observed with a strong dependence of conductivity on pH. Metagenomic and metatranscriptomic analysis showed that Desulfovibrio desulfuricans was the most dominant and metabolically active bacterium that contained and highly expressed the genes for e-pili. Abundance of genes encoding the total type IV pilus assembly proteins (6.72E-04 vs 1.24E-03, P < 0.05), PilA that determined the conductive properties (2.22E-04 vs 2.44E-04, P > 0.05), and PilB that proceeded the polymerization of pilin (1.56E-04 vs 3.52E-03, P < 0.05) with ethanol was lower than that with acetaldehyde. However, transcript abundance of these genes with ethanol was generally higher than that with acetaldehyde. In comparison to acetaldehyde, ethanol increased the transcript abundance of genes encoding the key enzymes involved in NADH/NAD+ transformation on complex I and ATP synthesis on complex V in intracellular electron transport chain. The improvement of intracellular electron transfer in D. desulfuricans suggested that electrons were intracellularly energized with high energy to activate e-pili during DIET.

NMRK2 is an efficient diagnostic indicator for Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinomas (tRCC) are a rare and highly malignant type of renal cancer, lacking efficient diagnostic indicators and therapeutic targets. Through the analysis of public databases and our cohort, we identified NMRK2 as a potential diagnostic marker for distinguishing Xp11.2 tRCC from kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) due to its specific upregulation in Xp11.2 tRCC tissues. Mechanistically, we discovered that TFE3 fusion protein binds to the promoter of the NMRK2 gene, leading to its upregulation. Importantly, we established RNA- and protein-based diagnostic methods for identifying Xp11.2 tRCC based on NMRK2 expression levels, and the diagnostic performance of our methods was comparable to a dual-color break-apart fluorescence in situ hybridization assay. Moreover, we successfully identified fresh Xp11.2 tRCC tissues after surgical excision using our diagnostic methods and established an immortalized Xp11.2 tRCC cell line for further research purposes. Functional studies revealed that NMRK2 promotes the progression of Xp11.2 tRCC by upregulating the NAD+/NADH ratio, and supplementation with ß-nicotinamide mononucleotide (NMN) or nicotinamide riboside chloride (NR), effectively rescued the phenotypes induced by the knockdown of NMRK2 in Xp11.2 tRCC. Taken together, these data introduce a new diagnostic indicator capable of accurately distinguishing Xp11.2 tRCC and highlight the possibility of developing novel targeted therapeutics. © 2024 The Pathological Society of Great Britain and Ireland.

Aged fragmented-polypropylene microplastics induced ageing statues-dependent bioenergetic imbalance and reductive stress: In vivo and liver organoids-based in vitro study.

Ageing is a nature process of microplastics that occurrs daily, and human beings are inevitably exposed to aged microplastics. However, a systematic understanding of ageing status and its toxic effect is currently still lacking. In this study, plastic cup lids-originated polypropylene (PP) microplastics were UV-photoaged until the carbonyl index (CI), a canonical indicator for plastic ageing, achieved 0.08, 0.17, 0.22 and 0.28. The adverse hepatic effect of these aged PPs (aPPs) was evaluated in Balb/c mice (75 ng/mL water, about 200 particles/day) and human-originated liver organoids (LOs, 50 particles/mL, ranged from 5.94 to 13.15 ng/mL) at low-dose equivalent to human exposure level. Low-dose of aged PP could induce hepatic reductive stress both in vitro and in vivo, by elevating the NADH/NAD+ratio in a CI-dependent manner, together with hepatoxicity (indicated by increased AST secretion and cytotoxicity), and disrupted the genes encoding the nutrients transporters and NADH subunits accompanied by the restricted ATP supply, declined mitochondrial membrane potential and mitochondrial complexI/IV activities, without significant increase in MDA levels in the liver. These changes in the liver disrupted systematic metabolism, representing a circulatory panel of increases in the lactate, triglyceride, Fgf21 levels, and decreases in the pyruvate level, linked the reductive stress to the declined body weight gain but elevated hepatic NADH contents following aPPs exposure. Additionally, assessing by the LOs, it was found that digestion drastically accelerated the ageing of aPPs and worsen the energy supply upon mitochondria, representing a "scattergun effect" induced by the formation of micro- and nano-plastics mixture toward NADH/NAD+imbalance.

Valacyclovir and Acyclovir Are Substrates of the Guanine Deaminase Cytosolic PSD-95 Interactor (Cypin).

Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither Km nor KD values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy.

Rewiring the reductive TCA pathway and glyoxylate shunt of Escherichia coli for succinate production from corn stover hydrolysate using a two-phase fermentation strategy.

Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.

Assaying D-Alanine Racemase in Lactic Acid Bacteria Using NADH Oxidoreduction Enzymic System.

A notable characteristic of amino acids is their optical isomerism, existing as L-form and D-form. Proteins are composed exclusively of L-form amino acids. However, recently, it is reported that D-alanine is evaluated particularly highly in terms of sensory evaluation. D-body amino acids convert L-body amino acid proteolysis from a substrate such as foods during fermentation of lactic acid bacteria. This chapter presents a description of methods used for D-alanine racemase assays in the solution producing by lactic acid bacteria (LAB) using D-amino acid oxidase and lactic acid dehydrogenase via a NADH oxidoreduction system.

Fluorescence Spectroscopy With Temperature Functional Tests in the Assessment of Markers of Intracellular Energy Metabolism: Spatial Heterogeneity and Reproducibility of Measurements.

The fluorescence intensities of the cellular respiratory cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD++ (oxidized flavin adenine dinucleotide) reflect energy metabolism in skin and other tissues and can be quantified in vivo by fluorescence spectroscopy (FS). However, the variability of physiological parameters largely determines the reproducibility of measurement results and the reliability of the diagnostic test. In this prospective study, we evaluated the interday reproducibility of NADH and FAD++ fluorescence intensity measurements in the skin of 51 healthy volunteers assessed by the FS at baseline, after local cooling (10°C) and heating of the skin (35°C). Results showed that the fluorescence amplitude of NADH (AFNADH) in forearm skin was the most reproducible of the FS parameters studied. Assessment of AFNADH in the dorsal forearm in combination with a thermal functional test is the most promising method for clinical use for assessing energy metabolism in the skin.

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol.

BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

Homeostatic regulation of NAD(H) and NADP(H) in cells.

Nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and nicotinamide adenine dinucleotide phosphate (NADP+)/reduced NADP+ (NADPH) are essential metabolites involved in multiple metabolic pathways and cellular processes. NAD+ and NADH redox couple plays a vital role in catabolic redox reactions, while NADPH is crucial for cellular anabolism and antioxidant responses. Maintaining NAD(H) and NADP(H) homeostasis is crucial for normal physiological activity and is tightly regulated through various mechanisms, such as biosynthesis, consumption, recycling, and conversion between NAD(H) and NADP(H). The conversions between NAD(H) and NADP(H) are controlled by NAD kinases (NADKs) and NADP(H) phosphatases [specifically, metazoan SpoT homolog-1 (MESH1) and nocturnin (NOCT)]. NADKs facilitate the synthesis of NADP+ from NAD+, while MESH1 and NOCT convert NADP(H) into NAD(H). In this review, we summarize the physiological roles of NAD(H) and NADP(H) and discuss the regulatory mechanisms governing NAD(H) and NADP(H) homeostasis in three key aspects: the transcriptional and posttranslational regulation of NADKs, the role of MESH1 and NOCT in maintaining NAD(H) and NADP(H) homeostasis, and the influence of the circadian clock on NAD(H) and NADP(H) homeostasis. In conclusion, NADKs, MESH1, and NOCT are integral to various cellular processes, regulating NAD(H) and NADP(H) homeostasis. Dysregulation of these enzymes results in various human diseases, such as cancers and metabolic disorders. Hence, strategies aiming to restore NAD(H) and NADP(H) homeostasis hold promise as novel therapeutic approaches for these diseases.

Formate-Mediated Electroenzymatic Synthesis via Biological Cofactor NADH.

Synthetic biohybrid systems by coupling artificial system with nature's machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate-driven NADH regeneration. Utilising a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90% Faraday efficiency at a current density of 150 mA cm-2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilised redox enzymes-alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and L-glutamate dehydrogenase (GDH)-to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.