Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

The effect of NAPRTase overexpression on the total levels of NAD,the NADH/NAD+ ratio,and the distribution of metabolites in Escherichia coli

Escherichia coli (E. coli) maintains its total NADH/NAD+ intracellular pool by synthesizing NAD through the de novo pathway and the pyridine nucleotide salvage pathway. The salvage pathway recycles intracellular NAD breakdown products and preformed pyridine compounds from the environment, such as nicotinic acid (NA). The enzyme nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11), encoded by the pncB gene, catalyzes the formation of nicotinate mononucleotide (NAMN), a direct precursor of NAD, from NA. This reaction is believed to be the rate-limiting step in the NAD salvage pathway. The current study investigates the effect of overexpressing the pncB gene from Salmonella typhimurium on the total levels of NAD, the NADH/NAD+ ratio, and the production of different metabolites in E. coli under anaerobic chemostat conditions and anaerobic tube experiments. In addition, this paper studies the effect of combining the overexpression of the pncB gene with an NADH regeneration strategy that increases intracellular NADH availability, as we have previously shown. (The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, 230-237; Metabolic engineering of Escherichia coli: Increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229.) Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, overexpression of the pncB gene led to a significant shift in the metabolic patterns as evidenced by a decrease in lactate production and an increase as high as two-fold in the ethanol-to-acetate (Et/Ac) ratio. These results suggest that under chemostat conditions the total level of NAD is not limiting and the metabolic rates are fixed by the system at steady state. On the other hand, under transient conditions (such as those in batch cultivation) the increase in the total level of NAD can increase the rate of NADH-dependent pathways (ethanol) and therefore change the final distribution of metabolites. The effect of combining overexpression of the pncB gene with the substitution of the native cofactor-independent formate dehydrogenase (FDH) with an NAD(+)-dependent FDH was also investigated under anaerobic tube conditions. This manipulation produced a metabolic pattern that combines a high Et/Ac ratio similar to that obtained with the new FDH with an intermediate lactate level similar to that obtained with the overexpression of the pncB gene. It was found that addition of the pncB gene to the FDH system does not increase further the production of reduced metabolites because the system for NADH regeneration already reached the maximum theoretical yield of approximately 4 mol NADH/mol of glucose.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

Role of NAD in regulating the adhE gene of Escherichia coli.

The fermentative alcohol dehydrogenase of Escherichia coli is encoded by the adhE gene, which is induced under anaerobic conditions but repressed in air. Previous work suggested that induction of adhE might depend on NADH levels. We therefore directly measured the NAD+ and NADH levels for cultures growing aerobically and anaerobically on a series of carbon sources whose metabolism generates different relative amounts of NADH. Expression of adhE was monitored both by assay of alcohol dehydrogenase activity and by expression of phi(adhE'-lacZ) gene fusions. The expression of the adhE gene correlated with the ratio of NADH to NAD+. The role of NADH in eliciting adhE induction was supported by a variety of treatments known to change the ratio of NADH to NAD+ or alter the total NAD+-plus-NADH pool. Blocking the electron transport chain, either by mutation or by chemical inhibitors, resulted in the artificial induction of the adhE gene under aerobic conditions. Conversely, limiting NAD synthesis, by introducing mutational blocks into the biosynthetic pathway for nicotinic acid, decreased the expression of adhE under anaerobic conditions. This, in turn, was reversed by supplementation with exogenous NAD or nicotinic acid. In merodiploid strains carrying deletion or insertion mutations abolishing the synthesis of AdhE protein, an adhE-lacZ fusion was expressed at nearly 10-fold the level observed in an adhE+ background. Introduction of mutant adhE alleles producing high levels of inactive AdhE protein gave results equivalent to those seen in absence of the AdhE protein. This finding implies that it is the buildup of NADH due to lack of enzyme activity, rather than the absence of the AdhE protein per se, which causes increased induction of the phi(adhE'-lacZ) fusion. Moreover, mutations giving elevated levels of active AdhE protein decreased the induction of the phi(adhE'-lacZ) fusion. This finding suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerobic conditions, thus indirectly regulating its own expression.     

Glyoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution

K562 erythroleukemic cells cultured at low population density in the absence of serum die within 12-24 hours, unless 0.1 mM glyoxylic acid is added to the culture medium. Earlier events, preceding cell death and occurring within 2 hours culture, are: a) a marked drop of both the NAD+/NADH ratio and the NAD+ concentration, which is prevented by 10mM benzamide, b) an increased biosynthesis of NAD+, leading to extensive depletion of cellular ATP. In the presence of 0.1 mM glyoxylic acid the NAD+/NADH ratio as well as their absolute concentrations remain unchanged, while NAD+ biosynthesis is absent. A NAD+/NADH glycohydrolase activity is present in the cell extract, inhibited by 10 mM benzamide and with a higher affinity for NADH than for NAD+. Preservation of a high NAD+/NADH ratio by glyoxylic acid apparently prevents enzyme activity and the related loss of pyridine nucleotides.     

Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy-scyllo-inosose with an aid of NAD(+)-NADH recycling. The NAD(+)-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K(i) values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD(+), the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4-(2)H]phosphonate and was found to be pro-R specific.     

Levels of Nicotinamide Adenine Dinucleotide and Reduced Nicotinamide Adenine Dinucleotide in Facultative Bacteria and the Effect of Oxygen

Nicotinamide adenine dinucleotide (NAD) and reduced NAD (NADH) levels have been measured in bacterial cultures. The cofactors were assayed by using the very sensitive cycling assay described previously by Cartier. Control experiments showed that the level of total NAD(H) falls during harvesting, and so samples were taken quickly from growing cultures and extracted immediately without separating the cells from the medium. Total NAD(H) ranged from 4.0 to 11.7 mumoles/g of dry cells for three facultative organisms, Klebsiella aerogenes, Escherichia coli, and Staphylococcus albus. NADH was remarkably constant in these bacteria; only one out of ten series of determinations was outside the range 1.4 to 1.9 mumoles/g of dry cells. NAD(+) showed much greater variation. An anaerobe (Clostridium welchii) had significantly more total NAD(H) whereas an aerobe Pseudomonas aeruginosa had about as much NAD(H) as the facultative organisms. NAD and NADH were measured during growth: once more NADH was much more constant than NAD. During change-over between aerobiosis and anaerobiosis, NADH showed a temporary increase but then returned to a constant level, whereas NAD changed from high aerobically to low anaerobically. These results are discussed in terms of the control mechanisms that may be involved.     

Regeneration of NAD(P)H by immobilized whole cells of Clostridium butyricum under hydrogen high pressure

Immobilized whole cells of Clostridium butyricum reduced both NAD(+) and NADP(+) in the presence of hydrogen at a pressure of 100 atm. The NAD(+) and NADP(+) reduction activities were 4.45 and 4.30 U/g dry cells, respectively [U = NAD(P)H regenerated, mu mol/min]. The amount of NADH regenerated by immobilized cells increased with increasing hydrogen pressure above 10 atm. Immobilized cells (6 mg dry cells) of Cl. butyricum completely converted NAD(+) (6.4 mumole) to NADH for 5 h, whereas only 60% of NAD(+) were reduced by free cells. Immobilized cells retained 89% activity after the 5-h reactions were repeated 4 times. L-Alanine was continuously produced at the rate of 12.8 mumol/min g dry cells from hydrogen, ammonium, and pyruvate with immobilized Cl. butyricum-alanine dehydrogenase.     

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

Kinetics of NADH oxidation of NAD+ reduction by mitochondrial complex I]

The kinetics of the NAD: artificial acceptor-oxidoreductase and delta mu H(+)-dependent succinate: NAD(+)-oxidoreductase reactions (reverse electron transfer) reactions catalyzed by the membrane-bound complex I was studied. The values of apparent rate constants of dissociation of complexes of the oxidized and reduced enzyme with NAD+ and NADH were determined. It was shown that the apparent affinity of NADH for the oxidized complex I is by nearly three orders of magnitude as high as that of the reduced one; a reverse correlation is found for NAD+. A kinetic scheme of complex I functioning in the forward and reverse reactions, according to which the free reduced enzyme is not an intermediate of the forward (NADH-oxidase) reaction and the free oxidized enzyme is not an intermediate of the reverse (NAD(+)-reductase) reaction, is proposed.     

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Inheritance and expression of NAD(P)H nitrate reductase in barley

Summary NADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.Scientific Paper No. 7617, College of Agriculture Research Center and Home Economics, Washington State University, Pullman, WA, USA. Project Nos. 0233 and 0745     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.     

Structure and function of NAD kinase and NADP phosphatase: key enzymes that regulate the intracellular balance of NAD(H) and NADP(H)

The functions of NAD(H) (NAD(+) and NADH) and NADP(H) (NADP(+) and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substrate-binding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.