Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO₂ rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD⁺ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.     

The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems

Malic enzyme (ME; NADP⁺-dependent; EC 1.1.40) provides NADPH for lipid biosynthesis in oleaginous microorganisms. Its role in vivo depends on there being an adequate supply of NADH to drive malate dehydrogenase to convert oxaloacetate to malate as a component of a cycle of three reactions: pyruvate → oxaloacetate → malate and, by the action of ME, back to pyruvate. However, the availability of cytosolic NADH is limited and, consequently, ancillary means of producing NADPH are necessary. Stoichiometries are given for the conversion of glucose to triacylglycerols involving ME with and without the reactions of the pentose phosphate pathway (PPP) as an additional source of NADPH. Some oleaginous microorganisms (such as Yarrowia lipolytica), however, lack a cytosolic ME and, if the PPP is the sole provider of NADPH, the theoretical yield of triacylglycerol from glucose falls to 27.6 % (w/w) from 31.6 % when ME is present. An alternative route for NADPH generation via a cytosolic isocitrate dehydrogenase (NADP⁺-dependent) is then discussed.     

Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation

A sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD⁺ or NADP⁺. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP⁺. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD⁺. Relative to nonproliferating cells, NAD⁺-linked malic enzyme activity was slightly reduced and the NADP⁺-linked activity was unchanged in the particulate fraction of serum-stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two-fold increase in the ratio of NAD(P)⁺-linked malic enzyme to NAD⁺-linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP⁺-dependent malic enzyme activity and a decrease in NAD⁺-linked malate dehydrogenase indictated an increase in the ratio of pyruvate-producing to oxalacetate-producing malate oxidase activity in the cytosol of proliterating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.     

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO₃⁻ or NH₄⁺ assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH₄⁺ was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO₃⁻ addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO₃⁻ assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO₃⁻ assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH₄⁺ assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO₃⁻ assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.     

Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD⁺, NADH) of NAD⁺-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD⁺ was the varied substrate. However, the NAD⁺-linked malic enzyme exhibited the weakest affinity for NAD⁺ and the lowest sensitivity for NADH. It is suggested that NAD⁺-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD⁺ addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD⁺ in potato mitochondria, which prove to take up added NAD⁺. This mechanism concentrates NAD⁺ in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD⁺/NADH ratio.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

Changes in the electron transport chain of pea leaf mitochondria metabolizing malate

Pea leaf mitochondria showed complex kinetics for malate metabolism. O₂ uptake increased as malate concentration increased from 0 to 10 mm, reached a plateau between 10 and 20 mm malate, and then increased again up to 40 mm malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O₂ uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O₂ uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O₂ uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing K_m values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 mm malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O₂ through the alternative oxidase. Although this coincided with the second phase of malate-dependent O₂ uptake it was not required for this phase to be seen.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.