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Abstract Background Hepatocellular carcinoma (HCC) is a principal type of liver cancer with high incidence and mortality rates. Regorafenib is a novel oral multikinase inhibitor for second-line therapy for advanced HCC. However, resistance to regorafenib is gradually becoming a dilemma for HCC and the mechanism remains unclear. In this study, we aimed to reveal the metabolic profiles of regorafenib-resistant cells and the key role and mechanism of the most relevant metabolic pathway in regorafenib resistance. Methods Metabolomics was performed to detect the metabolic alteration between drug-sensitive and regorafenib-resistant cells. Colony formation assay, CCK-8 assay and flow cytometry were applied to observe cell colony formation, cell proliferation and apoptosis, respectively. The protein and mRNA levels were detected by western blot and RT-qPCR. Cell lines of Glucose-6-phosphate dehydrogenase(G6PD) knockdown in regorafenib-resistant cells or G6PD overexpression in HCC cell lines were stably established by lentivirus infection technique. G6PD activity, NADPH level, NADPH/NADP+ ratio, the ratio of ROS positive cells, GSH level, and GSH/GSSG ratio were detected to evaluate the anti-oxidative stress ability of cells. Phosphorylation levels of NADK were evaluated by immunoprecipitation. Results Metabonomics analysis revealed that pentose phosphate pathway (PPP) was the most relevant metabolic pathway in regorafenib resistance in HCC. Compared with drug-sensitive cells, G6PD enzyme activity, NADPH level and NADPH/NADP+ ratio were increased in regorafenib-resistant cells, but the ratio of ROS positive cells and the apoptosis rate under the conditions of oxidative stress were decreased. Furthermore, G6PD suppression using shRNA or an inhibitor, sensitized regorafenib-resistant cells to regorafenib. In contrast, G6PD overexpression blunted the effects of regorafenib to drug-sensitive cells. Mechanistically, G6PD, the rate-limiting enzyme of PPP, regulated the PI3K/AKT activation. Furthermore, PI3K/AKT inhibition decreased G6PD protein expression, G6PD enzymatic activity and the capacity of PPP to anti-oxidative stress possibly by inhibited the expression and phosphorylation of NADK. Conclusion Taken together, a feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC and targeting the feedback loop could be a promising approach to overcome drug resistance.

Background: Hepatocellular carcinoma (HCC) is a principal type of liver cancer with high incidence and mortality rates. Regorafenib is a novel oral multikinase inhibitor for second-line therapy for advanced HCC. However, resistance to regorafenib is gradually becoming a dilemma for HCC and the mechanism remains unclear. In this study, we aimed to reveal the metabolic profiles of regorafenib-resistant cells and the key role and mechanism of the most relevant metabolic pathway in regorafenib resistance. Methods: Metabolomics was performed to detect the metabolic alteration between drug-sensitive and regorafenib-resistant cells. Colony formation assay, CCK-8 assay and flow cytometry were applied to observe cell colony formation, cell proliferation and apoptosis, respectively. The protein and mRNA levels were detected by western blot and RT-qPCR. Cell lines of Glucose-6-phosphate dehydrogenase(G6PD) knockdown in regorafenib-resistant cells or G6PD overexpression in HCC cell lines were stably established by lentivirus infection technique. G6PD activity, NADPH level, NADPH/NADP+ ratio, the ratio of ROS positive cells, GSH level, and GSH/GSSG ratio were detected to evaluate the anti-oxidative stress ability of cells. Phosphorylation levels of NADK were evaluated by immunoprecipitation. Results: Metabonomics analysis revealed that pentose phosphate pathway (PPP) was the most relevant metabolic pathway in regorafenib resistance in HCC. Compared with drug-sensitive cells, G6PD enzyme activity, NADPH level and NADPH/NADP+ ratio were increased in regorafenib-resistant cells, but the ratio of ROS positive cells and the apoptosis rate under the conditions of oxidative stress were decreased. Furthermore, G6PD suppression using shRNA or an inhibitor, sensitized regorafenib-resistant cells to regorafenib. In contrast, G6PD overexpression blunted the effects of regorafenib to drug-sensitive cells. Mechanistically, G6PD, the rate-limiting enzyme of PPP, regulated the PI3K/AKT activation. Furthermore, PI3K/AKT inhibition decreased G6PD protein expression, G6PD enzymatic activity and the capacity of PPP to anti-oxidative stress possibly by inhibited the expression and phosphorylation of NADK. Conclusion: Taken together, a feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC and targeting the feedback loop could be a promising approach to overcome drug resistance. [ABSTRACT FROM AUTHOR]

Arabidopsis NADK2 (NAD kinase 2) is a chloroplast-localized enzyme involved in NADP+ synthesis, which acts as the final electron acceptor in the photosynthetic electron transfer chain. The NADK2-deficient mutant (nadk2) was used to analyze the effect of NAD(P)(H) unbalance in the dark-induced leaf senescence. During senescence, WT plants and nadk2 mutants showed a similar reduction in chlorophyll content. NAD(P)(H) quantification showed that the amount of total NAD(P)(H) decreased on the day 7 in WT but on the day 3 in nadk2. The phosphorylation ratio (i.e. NADP(H)/NAD(H)) decreased on day 1 in WT. In contrast, the nadk2 showed lower phosphorylation ratio at 0 day and no change throughout the aging process. Metabolome analysis showed that the metabolic profiles of both WT plants and nadk2 mutants subjected to dark-induced senescence adopted similar patterns as the senescence progressed. However, the changes in individual metabolites in the nadk2 mutants were different from those of the WT during dark-induced senescence.

Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8 h light / 16 h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2−, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities. [ABSTRACT FROM AUTHOR]

In this study, 5 mM calcium acetate and calcium chloride were individually sprayed twice on rice leaves to study their function in antioxidant response under ozone stress (125 ± 25 ppb). Results showed that elevated O3 during rice cultivation impaired photosystem II (PSII) and carbon dioxide assimilation, as indicated by a decrease in effective quantum yield of PSII and electron transport rate and an increase in intracellular carbon dioxide. This occurrence led to growth inhibition resulting in rice yield and grain size (width and thickness) reduction, accompanied with the increase of malondialdehyde (MDA) content in plant cells under O3 stress. Rice plant pretreated with calcium acetate could endure long-term O3 exposure through the enhancement of NAD kinase activity and NADPH content, leading to an increase in ascorbate peroxidase (APX) and glutathione reductase (GR) activities and higher levels of reduced ascorbic acid (ASA) and reduced glutathione (GSH) under long-term O3 exposure. Consequently, the application of calcium acetate led to increase PSII efficiency and reduce cell damages (MDA content), resulting in an increase in rice yield and grain size under O3 stress. Under long-term O3 exposure, pretreatment of rice with calcium chloride resulted in a decrease in APX activity and an increase in MDA level, resulting in a rice yield similar to that of the non-pretreatment. It suggested that calcium acetate pretreatment of rice was more effective than pretreatment with calcium chloride in terms of reducing stress and maintaining antioxidant enzyme activities, and redox compounds in cells under long-term O3 exposure. [ABSTRACT FROM AUTHOR]

Global burden of infectious diseases and antimicrobial resistance are major public health issues calling for innovative control measures. Bacterial NAD kinase (NADK) is a crucial enzyme for production of NADP(H) and growth. In Staphylococcus aureus , NADK promotes pathogenesis by supporting production of key virulence determinants. Here, we find that knockdown of NADK by CRISPR interference sensitizes S. aureus to osmotic stress and to stresses induced by antibiotics targeting the envelop as well as replication, transcription and translation. Thus, NADK represents a promising target for the development of inhibitors which could be used in combination with current antibiotics. [ABSTRACT FROM AUTHOR]

The intracellular NADPH insufficient supply is the main bottleneck to the synthesis of chiral alcohols by asymmetric reduction with whole-cell catalysis. Herein, we provide a novel strategy to strengthen intracellular NADPH supply through introducing an NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (gapB from Bacillus subtilis 168) into the Embden-Meyerhof pathway and a NAD kinase (yfjB from E. coli MG1655) to further enhance the NADP(H) pool. A recombinant E. coli (E. coli BL21 (DE3)/pETDuet-1-gapB-yueD&pET28a-yfjB) was constructed to co-express gapB and yfjB with a carbonyl reductase gene yueD together. The result showed that the intracellular NADPH amount increased by 134.4% with the strategy. To the model reaction (asymmetric reduction of acetophenone to S-phenyl ethanol), the yield was 3.7-fold with this strategy compared to the control. This provides a technological route for strengthening the intracellular NADPH supply in E. coli for biocatalysis and biosynthesis. [ABSTRACT FROM AUTHOR]

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L -1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L -1  h -1 . Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals., (© 2024 Wiley‐VCH GmbH.)

NAD kinase (NADK) is required for the de novo synthesis of NADP+ from NAD+. In neutrophils, NADK plays an essential role by providing sufficient levels of NADPH to support a robust oxidative burst. Activation of NADPH oxidase-2 (NOX-2) in neutrophils by stimulators of protein kinase C (PKC), such as phorbol myristate acetate (PMA), results in the rapid generation of superoxide at the expense of oxidation of NADPH to NADP+. In this study, we measured the levels of pyridine nucleotides following the addition of PMA to neutrophils. PMA elicited a rapid increase in NADP+ in neutrophils, which was not due to oxidation of NADPH, the levels of which also rose. This was mirrored by a rapid reduction in NAD+ levels, suggesting that NADK had been activated. PMA-induced depletion of NAD+ in neutrophils was blocked by PKC inhibitors, but was not dependent on NOX-2, as it was not blocked by the NOX inhibitor, diphenyleneiodonium. PMA also increased NADK activity in neutrophil lysates as well as NADK phosphorylation, as revealed by a monoclonal antibody selective for phospho-NADK. Human recombinant NADK was phosphorylated by PKCδ, resulting in increased immunoreactivity, but unchanged enzyme activity, suggesting that PKC-induced phosphorylation alone is insufficient to increase NADK activity in neutrophils. This leads us to speculate that phosphorylation of NADK promotes the dissociation of an inhibitory molecule from a complex, thereby increasing enzyme activity. Activation of NADK by PKC in phagocytic cells could be critical for the rapid provision of sufficient levels of superoxide for host defence against invading microorganisms. Image 1 • Phorbol myristate acetate increases NADP and NADPH in neutrophils. • The mechanism for this effect is activation of NAD kinase. • NAD kinase in neutrophils is activated by protein kinase C-induced phosphorylation. • Phosphorylation of NAD kinase alone is insufficient to increase enzyme activity. • Phosphorylation might induce the release of an inhibitory protein from NAD kinase. [ABSTRACT FROM AUTHOR]

Background: Amoebiasis is caused by Entamoeba histolytica, which is a pathogenic species living on human colon tissues. The development of new drugs for anti-amebic are still very needed for clinical treatment. Objective: This aims to identify the compounds in Cratoxylum sumatranum for their anti-amoeba activity. Materials and Methods: In this study we used bioactivity-guided isolation and structural analysis to identified anti-amebic compounds from dichloromethane extract of Cratoxylum sumatranum stem bark. Their anti-amebic activity was determined by an in vitro cell-based assay against Entamoeba histolytica and an enzymatic assay on NAD kinase. Results: Two known compounds from the cage xanthone groups, namely cochinchinoxanthone (1) and cochinchinone D (2), were isolated. The structures of the cage xanthone compounds were established by extensive spectroscopic data analysis. Compound (1) showed the greatest level of anti-amebic activity both in cell-based and enzymatic assay, yielding IC50 values of 4.57 and 12.17 μg/mL, respectively. In contrast, compound (2) yielded IC50 values of 5.19 and 12.60 μg/mL, respectively. Conclusion: When considering the demonstrated anti-amebic activities, it becomes apparent that these compounds, isolated from Cratoxylum sumatranum stem bark, have the potential to be further developed into effective anti-amebic medicine against Entamoeba histolytica. [ABSTRACT FROM AUTHOR]

Sulfur is essential for all living organisms due to its ability to mediate a variety of enzymatic reactions, signalling networks, and redox processes. The interplay between sulfhydryl group (SH) and disulfide bond (S–S) is central to the maintenance of intracellular oxidative balance. Although most aerobic organisms succumb to sulfur starvation, the nutritionally versatile soil microbe Pseudomonas fluorescens elaborates an intricate metabolic reprogramming in order to adapt to this challenge. When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. While oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle, metabolic modules known to generate reactive oxygen species are impeded, the activities NADPH-producing enzymes such as malic enzyme, and glutamate dehydrogenase (GDH) NADP-dependent are increased. The α-ketoglutarate (KG) generated from glutamine rapidly enters the TCA cycle via α-ketoglutarate dehydrogenase (KGDH), an enzyme that was prominent in the control cultures. In the S-deficient media, the severely impeded KGDH coupled with the increased activity of the reversible isocitrate dehydrogenase (ICDH) that fixes KG into isocitrate in the presence of NADH and HCO3− ensures a constant supply of this critical tricarboxylic acid. The up-regulation of ICDH-NADP dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis, a reaction aided by the concomitant increase in NAD kinase activity. The latter converts NAD into NADP in the presence of ATP. Taken together, the data point to a metabolic network involving isocitrate, α-KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment. [ABSTRACT FROM AUTHOR]

The use of oxidoreductases as biocatalysts for industrial production of valuable compounds has a strong demand for NADP. Herein, we prepared superparamagnetic NAD kinase catalyst to synthesize NADP in vitro. First, Fe3O4 particles were synthesized through a solvothermal method, followed by the chemical modification with epichloro-hydrin, iminodiacetic acid, and Ni2+ to yield functional Fe3O4 sub-microspheres. Subsequently, NAD kinase of Escherichia coli was overexpressed and immobilized on to the surface of magnetic sub-microspheres. The immobilized NAD kinase was used to catalyze the conversion of NAD to NADP in a cell-free system. Under optimal condition, the conversion ratio of NAD reached 91.7% and remained at 86.3% after repeated use for five times. Our study revealed that the novel magnetic NAD kinase catalyst possessed favorable properties for magnetic manipulation and NADP production. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) signaling and nicotinamide adenine dinucleotide (NAD) signaling are two basic signal regulation pathways in organisms, playing crucial roles in signal transduction, energy metabolism, stress tolerance, and various developmental processes. Notably, calmodulins (CaMs) and NAD kinases (NADKs) are important hubs for connecting these two types of signaling networks, where CaMs are the unique activators of NADKs. NADK is a key enzyme for NADP (including NADP+ and NADPH) biosynthesis by phosphorylating NAD (including NAD+ and NADH) and therefore, maintains the balance between NAD pool and NADP pool through an allosteric regulation mode. In addition, the two respective derivatives from NAD+ (substrate of NADK) and NADP+ (product of NADK), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), have been considered to be the important messengers for intracellular Ca2+ homeostasis which could finally influence the combination between CaM and NADK, forming a feedback regulation mechanism. In this review article, we briefly summarized the major research advances related to the feedback regulation pathway, which is activated by the interaction of CaM and NADK during plant development and signaling. The theories and fact will lay a solid foundation for further studies related to CaM and NADK and their regulatory mechanisms as well as the NADK-mediated NAD signaling behavior in plant development and response to stress.

Unicellular cyanobacteria are thought to be the evolutionary ancestors of plant chloroplasts and are widely used both for chemical production and as model organisms in studies of photosynthesis. Although most research focused on increasing reducing power (that is, NADPH) as target of metabolic engineering, the physiological roles of NAD(P)(H) in cyanobacteria poorly understood. In cyanobacteria such as the model species Synechocystis sp. PCC 6803, most metabolic pathways share a single compartment. This complex metabolism raises the question of how cyanobacteria control the amounts of the redox pairs NADH/NAD+ and NADPH/NADP+ in the cyanobacterial metabolic pathways. For example, photosynthetic and respiratory electron transport chains share several redox components in the thylakoid lumen, including plastoquinone, cytochrome b6f (cyt b6f), and the redox carriers plastocyanin and cytochrome c6. In the case of photosynthesis, NADP+ acts as an important electron mediator on the acceptor-side of photosystem I (PSI) in the linear electron chain as well as in the plant chloroplast. Meanwhile, in respiration, most electrons derived from NADPH and NADH are transferred by NAD(P)H dehydrogenases. Therefore, it is expected that Synechocystis employs unique NAD(P)(H) -pool control mechanisms to regulate the mixed metabolic systems involved in photosynthesis and respiration. This review article summarizes the current state of knowledge of NAD(P)(H) metabolism in Synechocystis. In particular, we focus on the physiological function in Synechocystis of NAD kinase, the enzyme that phosphorylates NAD+ to NADP+.

Unicellular cyanobacteria are thought to be the evolutionary ancestors of plant chloroplasts and are widely used both for chemical production and as model organisms in studies of photosynthesis. Although most research focused on increasing reducing power (that is, NADPH) as target of metabolic engineering, the physiological roles of NAD(P)(H) in cyanobacteria poorly understood. In cyanobacteria such as the model species Synechocystis sp. PCC 6803, most metabolic pathways share a single compartment. This complex metabolism raises the question of how cyanobacteria control the amounts of the redox pairs NADH/NAD+ and NADPH/NADP+ in the cyanobacterial metabolic pathways. For example, photosynthetic and respiratory electron transport chains share several redox components in the thylakoid lumen, including plastoquinone, cytochrome b6f (cyt b6f), and the redox carriers plastocyanin and cytochrome c6. In the case of photosynthesis, NADP+ acts as an important electron mediator on the acceptor-side of photosystem I (PSI) in the linear electron chain as well as in the plant chloroplast. Meanwhile, in respiration, most electrons derived from NADPH and NADH are transferred by NAD(P)H dehydrogenases. Therefore, it is expected that Synechocystis employs unique NAD(P)(H) -pool control mechanisms to regulate the mixed metabolic systems involved in photosynthesis and respiration. This review article summarizes the current state of knowledge of NAD(P)(H) metabolism in Synechocystis. In particular, we focus on the physiological function in Synechocystis of NAD kinase, the enzyme that phosphorylates NAD+ to NADP+. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) signaling and nicotinamide adenine dinucleotide (NAD) signaling are two basic signal regulation pathways in organisms, playing crucial roles in signal transduction, energy metabolism, stress tolerance, and various developmental processes. Notably, calmodulins (CaMs) and NAD kinases (NADKs) are important hubs for connecting these two types of signaling networks, where CaMs are the unique activators of NADKs. NADK is a key enzyme for NADP (including NADP+ and NADPH) biosynthesis by phosphorylating NAD (including NAD+ and NADH) and therefore, maintains the balance between NAD pool and NADP pool through an allosteric regulation mode. In addition, the two respective derivatives from NAD+ (substrate of NADK) and NADP+ (product of NADK), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), have been considered to be the important messengers for intracellular Ca2+ homeostasis which could finally influence the combination between CaM and NADK, forming a feedback regulation mechanism. In this review article, we briefly summarized the major research advances related to the feedback regulation pathway, which is activated by the interaction of CaM and NADK during plant development and signaling. The theories and fact will lay a solid foundation for further studies related to CaM and NADK and their regulatory mechanisms as well as the NADK-mediated NAD signaling behavior in plant development and response to stress. [ABSTRACT FROM AUTHOR]

Summary: Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild‐type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real‐time reverse transcription PCR (qRT‐PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis‐related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose‐6‐phosphate and 6‐phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism. Significance Statement: Synechocystis sp. PCC 6803 contains two genes (sll1415 and slr0400) that encode proteins with NADK homology, but the biological function of these genes has not been revealed to date. Here, we show that the sll1415‐encoded NAD kinase is required to ensure an NADP+ supply sufficient for catabolism of glucose via the oxidative pentose phosphate pathway under photoheterotrophic conditions. [ABSTRACT FROM AUTHOR]

Summary: NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP+) and pool size (sum of NADPH and NADP+), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP+ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP+ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) kinases are essential and ubiquitous enzymes involved in the tight regulation of NAD/nicotinamide adenine dinucleotide phosphate (NADP) levels in many metabolic pathways. Consequently, they represent promising therapeutic targets in cancer and antibacterial treatments. We previously reported diadenosine derivatives as NAD kinase inhibitors with bactericidal activities on Staphylococcus aureus. Among them, one compound (namely NKI1) was found effective in vivo in a mouse infection model. With the aim to gain detailed knowledge about the selectivity and mechanism of action of this lead compound, we planned to develop a chemical probe that could be used in affinity-based chemoproteomic approaches. Here, we describe the first functionalized chemical probe targeting a bacterial NAD kinase. Aminoalkyl functional groups were introduced on NKI1 for further covalent coupling to an activated SepharoseTM matrix. Inhibitory properties of functionalized NKI1 derivatives together with X-ray characterization of their complexes with the NAD kinase led to identify candidate compounds that are amenable to covalent coupling to a matrix.

Metastasis is a significant cause of high mortality in lung cancer. Lymph node (LN) metastasis is the most common metastatic pathway in non-small cell lung cancer and the most crucial factor affecting the prognosis of NSCLC. Nevertheless, the molecular mechanism underlying metastasis is unknown. We demonstrated that higher NADK expression suggests worsened survival prognosis, and NADK expression positively correlates with the lymph node metastasis rate and TNM and AJCC stages in NSCLC patients. Moreover, patients with LN metastasis show higher NADK expression than those without LN metastasis. NADK can promote NSCLC progression by enhancing the migration, invasion, lymph node metastasis and growth of NSCLC cells. Mechanistically, NADK inhibits the ubiquitination and degradation of BMPR1A by interacting with Smurf1, further activating the BMPs signalling pathway and promoting ID1 transcription. In conclusion, NADK may be a potential diagnostic indicator and a novel therapeutic target for metastatic NSCLC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Nicotinamide adenine dinucleotides (NAD+ and NADP+) are electron mediators involved in various metabolic pathways. NADP(H) are produced by NAD kinase (NADK) through the phosphorylation of NAD(H). The Arabidopsis NADK3 (AtNADK3) is reported to preferentially phosphorylate NADH to NADPH and is localized in the peroxisome. To elucidate the biological function of AtNADK3 in Arabidopsis, we compared metabolites of nadk1 , nadk2 and nadk3 Arabidopsis T-DNA inserted mutants. Metabolome analysis revealed that glycine and serine, which are intermediate metabolites of photorespiration, both increased in the nadk3 mutants. Plants grown for 6 weeks under short-day conditions showed increased NAD(H), indicating a decrease in the phosphorylation ratio in the NAD(P)(H) equilibrium. Furthermore, high CO 2 (0.15%) treatment induced a decrease in glycine and serine in nadk3 mutants. The nadk3 showed a significant decrease in post-illumination CO 2 burst, suggesting that the photorespiratory flux was disrupted in the nadk3 mutant. In addition, an increase in CO 2 compensation points and a decrease in CO 2 assimilation rate were observed in the nadk3 mutants. These results indicate that the lack of AtNADK3 causes a disruption in the intracellular metabolism, such as in amino acid synthesis and photorespiration. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide kinases (NAD kinases) are essential and ubiquitous enzymes involved in the production of NADP(H) which is an essential cofactor in many metabolic pathways. Targeting NAD kinase (NADK), a rate limiting enzyme of NADP biosynthesis pathway, represents a new promising approach to treat bacterial infections. Previously, we have produced the first NADK inhibitor active against staphylococcal infection. From this linear di-adenosine derivative, namely NKI1, we designed macrocyclic analogues. Here, we describe the synthesis and evaluation of an original series of cyclic diadenosine derivatives as NADK inhibitors of two pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The nature and length of the link between the two adenosine units were examined leading to sub-micromolar inhibitors of NADK1 from L. monocytogenes , including its most potent in vitro inhibitor reported so far (with a 300-fold improvement compared to NKI1). [Display omitted] • A series of original diadenosine-based macrocycles as NADK inhibitor were developed. • Cyclic compounds displayed higher NADK inhibitory potency than acyclic precursors. • Crystal structures explain the affinity gain by intramolecular interactions. • Lm NADK1 inhibition by MC1 represents a 300-fold improvement in comparison to our lead NKI1. • High NADK inhibitory potency of macrocycles was not translated into antibacterial activity. [ABSTRACT FROM AUTHOR]

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus , in the micromolar range. They behave as NAD mimics with the 5′,5'-diphosphate group substituted by a 8,5′ thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5′ position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics. [ABSTRACT FROM AUTHOR]

With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli ( E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions. [ABSTRACT FROM AUTHOR]

NAD kinase (NADK) phosphorylates NAD(H) to NADP(H). The enzyme has a crucial role in the regulation of the NADP(H)/NAD(H) ratio in various organisms. The unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two NADK-encoding genes, sll1415 and slr0400 . To elucidate the metabolic change in NADK-deficient mutants growing under photoautotrophic conditions, we conducted metabolomic analysis using capillary electrophoresis mass spectrometry (CE-MS). The growth curves of the wild-type parent (WT) and NADK-deficient mutants (Δ1415 and Δ0400) did not show any differences under photoautotrophic conditions. The NAD(P)(H) balance showed abnormality in both mutants. However, only the metabolite pattern of Δ0400 showed differences compared to WT. These results indicated that the two NADK isoforms have distinct functions in cyanobacterial metabolism. [ABSTRACT FROM AUTHOR]

Background: Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β- hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5. Results: C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain. Conclusions: Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine. [ABSTRACT FROM AUTHOR]

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP + . Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. [ABSTRACT FROM AUTHOR]

Background:Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK) and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. Materials and Methods: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. Results: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814) revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Š observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. Conclusion: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.

The expression and purification of large recombinant proteins or protein complexes is problematic for some biotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biological characterization or reach commercialization, when large proteins or protein complexes are expressed in E. coli via the popular T7-based plasmid-driven expression system. There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of its drawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copy number, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, such as gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter of debate. Plant plastidial NAD kinases (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23) are a classic example of proteins with high molecular weight, that are difficult to express and purify with traditional T7-based technology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thaliana NADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-free chromosomal modifications. Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein than classic T7 expression systems, as well as better production, thus confirming that expression from one single chromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and applied research. • Chromosomal-driven expression systems improve bacterial growth after IPTG induction compared to plasmid expression systems. • Chromosomal expression successfully leads to production of the large and complex A. thaliana NADK2 protein in E. coli. • The recovery activity of NADK2 is improved by chromosomal-driven expression. [ABSTRACT FROM AUTHOR]

Objectives: With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production. Results: The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l from 18.2 g glucose l, while the wild type consumed 15.7 g glucose l to afford 2.34 g PHB l in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production. Conclusion: A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD+ kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin)mediates direct catalytic activation of NADK by Ca2+/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Background Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production. Results The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroGfbr, aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol. Conclusion An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production. [ABSTRACT FROM AUTHOR]

Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD +-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichiacoli was replaced with a NADP +-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP + availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD +-dependent GapA activity with NADP +-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds. [ABSTRACT FROM AUTHOR]

Abstract: Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic isobutanol production. This strategy will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. In addition, combined activation of PntAB and YfjB led to 28% and 22% increase of aerobic isobutanol titer and yield, resulting in production of 10.8g/L isobutanol in 24h with a yield of 0.62mol/mol. [Copyright &y& Elsevier]

Purpose: To improve the biosynthesis of L-arginine by overexpressing homologous NAD kinase (ppnk) in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) and low oxygen supply (LOS). Methods: A recombinant plasmid (pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk) was constructed in a shuttle vector pJC1 and transferred in L-arginine producing strain Corynebacterium crenatum SYPA5-5. Furthermore, fermentation was performed by shake flask method with consecutive determination of cell growth and glucose concentration. NAD+ kinase activity was studied by stop method and NADP(H) concentrations were determined by spectrophotometric enzymatic cycling method. To check the biosynthesis of amino acids, HPLC method was used to determine extracellular amino acid concentrations. Results: In HOS condition, NAD+ kinase activity increased by 116 %, while intracellular concentrations of NADP+ and NADPH increased by 7.3 and 36.8 %, respectively. Whereas, in LOS condition, NAD+ kinase activity increased 49%, with intracellular 14.67 and 15 % increases in NADP+ and NADPH respectively. More importantly, recombinant strain could produce 26.47 and 11.36 g/L L-arginine in HOS and LOS respectively, which is higher than control strain value of 24.29 and 7.58 g/L respectively. Conclusion: These results suggest that altering the concentration of co-enzymes by NAD kinase in Corynebacterium crenatum is an effective way to increase NADP+ with concurrent production of NADPH for further enhanced L-arginine biosynthesis in Corynebacterium crenatum in both conditions of high and low oxygen supply. [ABSTRACT FROM AUTHOR]

Abstract: NADPH is the key cofactor in l-isoleucine (Ile) biosynthetic pathway. To increase the Ile biosynthesis in Corynebacterium glutamicum ssp. lactofermentum JHI3-156, NADPH supply needs to be enhanced. Here NAD kinase, the key enzyme for the de novo biosynthesis of NADP+ and NADPH, were cloned and expressed in JHI3-156, and their influences on Ile production were analysed. Meanwhile, enzyme properties of NAD kinase from JHI3-156 (CljPpnK) were compared with that from C. glutamicum ssp. lactofermentum ATCC 13869 (ClPpnK). Four variations existed between CljPpnK and ClPpnK. Both PpnKs were poly(P)/ATP-dependent NAD kinases that used ATP as the preferred phosphoryl donor and NAD+ as the preferred acceptor. CljPpnK exhibited a higher activity and stability than ClPpnK and less sensitivity towards the effectors NADPH, NADP+, and NADH, partly due to the variations between them. The S57P variation decreased their activity. Expression of CljppnK and ClppnK in JHI3-156 increased the ATP-NAD+ kinase activity by 69- and 47-fold, respectively, the intracellular NADP+ concentration by 36% and 101%, respectively, the NADPH concentration by 95% and 42%, respectively, and Ile production by 37% and 24%, respectively. These results suggest that overexpressing NAD kinase is a useful metabolic engineering strategy to improve NADPH supply and isoleucine biosynthesis. [Copyright &y& Elsevier]

Succinate semialdehyde (SSA) is a mitochondrially generated intermediate in the metabolism of γ-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to γ-hydroxybutyrate (GHB) in plants exposed to various abiotic stress conditions. Recent evidence indicates that distinct cytosolic and plastidial glyoxylate reductase isoforms from Arabidopsis thaliana (L.) Heynh (GLYR1 and GLYR2, respectively) catalyze the in vitro conversion of SSA to GHB, as well as glyoxylate to glycolate, via NADPH-dependent reactions. In the present study, recombinant Arabidopsis GLYR1 was demonstrated to catalyze the NADPH-dependent reduction of both glyoxylate and SSA simultaneously to glycolate and GHB, respectively. Six-hour time-course experiments with intact vegetative wild-type Arabidopisis plants subjected to submergence demonstrated that GHB accumulates in rosette leaves, and this is accompanied by increasing levels of GABA and alanine, NADH/NAD+ and NADPH/NADP+ ratios, and GLYR1 and GLYR2 transcript abundance. The use of GLYR ( glyr1 or glyr2 knockout) and NAD kinase1 ( NADK1 suppression or overexpression) mutants demonstrated that under submergence the production of GHB is mediated via both GLYR isoforms, the loss of either GLYR1 or GLYR2 activity influences redox status and the levels of GABA and alanine, and the manipulation of NADP(H) availability, specifically in the cytosol, influences the production of GHB. These results suggest that biochemical mechanisms are more important than transcriptional mechanisms in the regulation of GLYR activity and SSA detoxification in plants during the onset of submergence-induced oxygen deficiency. [ABSTRACT FROM AUTHOR]

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP-biosynthetic enzyme that catalyzes phosphorylation of NAD to yield NADP using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, ), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP, on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP. [ABSTRACT FROM AUTHOR]

The synthesis of metabolically stable methylenebis(phosphonate) analogues of 2-, 4-, and 6-pyridones of nicotinamide adenine dinucleotide (NAD) is reported. In contrast to natural pyrophosphates, these NAD analogues are able to penetrate the cell membrane and can be used as probes in cellular assays. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform ( NbG6PDH- Cyto) and two plastidic isoforms ( NbG6PDH- P1 and NbG6PDH- P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH- P2-silenced plants, but not in NbG6PDH- Cyto- and NbG6PDH- P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH- P2 reduced HR cell death and ROS to levels near those in NbG6PDH- P2-silenced plants. Similarly, silencing NbG6PDH- P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway. [ABSTRACT FROM AUTHOR]

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca signals. Previously, we reported divergent soybean ( Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca-dependent electrophoretic mobility shift and Ca binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 ± 0.006 mol lysine (mol glucose)−1, which was 12% higher than that of the empty vector control strain. [ABSTRACT FROM AUTHOR]