Cyanide--an uncompetitive inhibitor of NAD(P)-dependent malic enzyme from human term placental mitochondria

The activity of NAD(P)-dependent mitochondrial malic enzyme was considerably inhibited by KCN, whereas under the same conditions azide affected the enzyme only slightly. Kinetic studies showed that KCN is an uncompetitive inhibitor of mitochondrial malic enzyme from human placenta. In contrast to the mitochondrial enzyme, the cytosolic malic enzyme was only slightly affected by KCN and under the same conditions the effect of azide was negligible. The effect of KCN was compared to this on the malic enzyme from other sources.     

Study of redox potential in cytochrome <Emphasis Type="Italic">c</Emphasis> covalently bound to terminal oxidase of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> FTU

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Differential characteristics of the cytosol and mitochondrial isozymes of malic enzyme from bovine brain. Effects of dicarboxylic acids and sulfhydryl reagents

The cytosol and mitochondrial isozymes of bovine brain malic enzyme were studied with respect to their sensitivity towards a series of dicarboxylic acids and sulfhydryl reagents. While no effects were obtained with the dicarboxylic acids in the case of the cytosol enzyme, the activity of the mitochondrial variant was increased considerably when either succinate, 2-mercaptosuccinate, or l-aspartate were tested at low concentrations of l-malate. The activation was associated with a clear decrease in the Hill coefficient for l-malate, and this has been taken as an indication of the presence of an allosteric site on the mitochondrial enzyme. The presence of l-malate or a dicarboxylate anion analog is required at this site in order to achieve optimal velocity. The activators were also effective in increasing the reductive carboxylation of pyruvate by the mitochondrial enzyme and had no effect on the cytosol variant. The two isozymes also showed a clear differential sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) and Hg²⁺, since the mitochondrial malic enzyme was inhibited by concentrations of these reagents far below those required in order to achieve an effect on the activity of the malic enzyme found in the cytosol.     

Mitochondrial Malic Enzyme: Purification from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization in Neurons of Rat Brain

Abstract: To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither crossreactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion-fixed rat brain, considerable resolution was achieved at the light-microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.     

Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

Exposure of mitochondria to oxidative stress and elevated Ca²⁺ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 h later. Respiring mitochondria actively accumulated added Ca²⁺, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca²⁺ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.     

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO₂ via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. -Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.These data were presented in part at the meeting of the American Society for Neurochemistry in Richmond, Virginia, March 1993     

Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.     

Redox equilibria in hydroxylamine oxidoreductase. Electrostatic control of electron redistribution in multielectron oxidative processes

We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK(a) values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c(554) results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule.     

Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme.

1. The component fatty acids of the endogenous phospholipids of microsomal preparations of Mucor, when shaken at 30 degrees C, increased in both chain length and in degree of unsaturation. The net effect was the production of gamma-linolenic acid which, over 2 h, increased from 17% to 32% of total fatty acids present. No further significant changes occurred after this time. 2. The major site for desaturation/elongation reactions was at the sn-2 position of PtdIns. PtdCho and PtdEtn were not implicated. 3. Of numerous metabolites and cofactors added to the microsomes, only malate could prolong the elongation/desaturation reactions for up to 6 h. This effect was shown to be due to a membrane-associated malic enzyme [malate dehydrogenase (decarboxylating) NADP+] with the NADPH produced being used in fatty-acid desaturation. 4. Kinetic analysis of cytosolic and microsomal enzymes [both in 0.1% (mass/vol.) Chaps] could not distinguish between them. However, when the microsomal malic enzyme was dialysed to remove Chaps, it lost 90% of activity, although the cytosolic malic enzyme lost only 20% activity. 5. The structural analogue of malate, tartronic acid, which is an inhibitor of malic enzyme, also inhibited the malate-induced stimulation of fatty-acyl group desaturation and elongation in the microsomal membranes. 6. It is concluded that two distinct malic enzymes exist, one soluble and one membrane bound, with similar active sites. Both have different roles in the production of NADPH, for lipid metabolism. The former will produce NADPH for fatty-acid biosynthesis whilst the latter produces NADPH for fatty-acid desaturation.     

Study of properties of NADP malate dehydrogenase from corn leaves]

Kinetic properties of purified chloroplast isoenzyme of the "malic" enzyme from corn leaves were studied. The enzyme had optimum activity at pH 8.0 and 36 degrees C. Under standart conditions the Michaelis constants for the "malic" enzyme with Mn2+ as cofactor are 0.091 mM for malate and 0.04 mM for NADP. In case of Mg2+ as cofactor they are 0.66 and 0.02 mM respectively. Respective Km values for the cofactors Mn2+ and Mg2+ are 0.018 and 0.091 mM. The activity of the "malic" enzyme was inhibited by reduced NADP and NAD, ATP, ADP, fructose-1,6-diphosphate, oxaloacetic, oxalic, glyoxylic, glycolic and alpha-ketoglutaric acids, as well as by phosphate anions and pyrophosphate. The inhibitory effect of all metabolites and ions is more pronounced in case of Mn, rather than Mg, used as cofactors for the reaction. A possibility of metabolic regulation of NADP-"malic" enzyme activity in the leaves of C4-plants, is discussed.     

Genetic variation of the cytoplasmic and mitochondrial malic enzymes in the monkey Macaca nemestrina

Electrophoretic variation of both the cytoplasmic and mitochondrial forms of the malic enzyme is described in Macaca nemestrina. Pedigree analysis of the observed phenotypes demonstrates that the two subcellular forms of the malic enzyme are genetically independent. The identity of the electrophoretic phenotypes in brain, heart muscle, liver, kidney, adrenal, and spleen from any given individual shows that each subcellular form is determined by the same genetic locus in a wide variety of tissues. After separation by ion exchange chromatography, the cytoplasmic and mitochondrial malic enzymes were shown to be distinct in their heat stability and K _m for malate, but no significant differences were found among the variants of the cytoplasmic enzyme or among the variants of the mitochondrial enzyme. It is possible that the polymorphism of the mitochondrial malic enzyme is selectively neutral.This study was supported by grant GM-15253 from the National Institutes of Health. One of us (G.S.O.) was a Special Fellow, U.S. Public Health Service (5F3 HD 43, 122-02); Fellow, National Genetics Foundation.     

Effect of Anaplerotic Pathways Activation on CO<Subscript>2</Subscript>-dependent Anaerobic Glucose Utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains Deficient in the Main Pathways of Mixed Acid Fermentation

The effect of anaplerotic pathways activation on CO₂-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO₂ generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO₂. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO₂ formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO₂ source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO₂-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle.     

An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions

The NAD- and NADP-dependent malic enzymes from rat liver and adrenal mitochondrial fractions were separated and partially purified by gel filtration on Sepharose 6B. Two activity peaks were observed. The first contained a malic enzyme capable of reducing either NAD or NADP. This enzyme showed sigmoid kinetics in plots of activity versus the malate concentration. Succinate was an allosteric activator and ATP was a competitive inhibitor of malate. The second peak showed hyperbolic kinetics in plots of activity versus the malate concentration and was unaffected by either succinate or ATP. The relative activities of the two malic enzymes were quite constant in the adrenal mitochondrial fractions. In the liver mitochondrial fractions, the activity of the first peak varied and was sometimes absent while the activity of the second peak was quite constant. The kinetic properties of the first malic enzyme implicate it as an important regulator of malate oxidation.     

Strain variation in Hymenolepis diminuta: enzyme profiles

In comparative study of respiratory metabolism, it was established that the relative proportions of respiratory end-products (succinic, acetic and lactic acids) differed consistently in two strains of Hymenolepis diminuta (Toronto and ANU). The ANU strain produced more lactic acid and less succinic acid under aerobic and anaerobic conditions. In the shift from aerobic to anaerobic conditions both strains compensated by increasing their outputs of succinic acid. The ANU strain possessed significantly higher activities of hexokinase, pyruvate kinase, lactate dehydrogenase, cytosolic and mitochondrial malic enzyme and cytosolic α-glycerophosphate dehy drogenase. The Toronto strain had significantly higher activities of fumarase, succinate dehydrogenase, and fumarate reductase. There were no significant differences in the activities of phosphoenolpyruvate carboxykinase and malic dehydrogenase between strains. The fumarase activity in the Toronto strain was 16 times that of the ANU strain, its K_m (malate) was 0.8mM, as opposed to 2.5 mM, and it was less sensitive to inhibition by NAD or ATP. These observations are consistent with the patterns of end-product formation in the two strains. Ratios of end-products and calculations of approximate redox balance suggest that the Toronto strain may have a greater capacity for aerobic metabolism.     

构建可合成非天然辅酶的圆红冬孢酵母工程菌*

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)及其还原态是生物体通用的氧化还原辅酶和重要小分子,参与胞内众多代谢反应,因此调控NAD水平不仅难以选择性作用于代谢途径,还常常产生意外的生物学效应。最近研究发现利用非天然辅酶烟酰胺胞嘧啶二核苷酸(nicotinamide cytosine dinucleotide,NCD),可构建正交的氧化还原催化体系,为调控胞内代谢提供了新机遇。为实现在产油酵母圆红冬孢酵母中建立NCD介导的氧化还原代谢,采用农杆菌介导转化方法,在基因组整合表达密码子优化的NCD合酶(NcdS)编码基因NCDS,获得系列有效表达NcdS的工程菌株。酶偶联法分析发现,工程菌细胞裂解液NcdS酶活达8.1×10^-3 U/OD_{600 nm}。通过高效液相色谱法(HPLC)和超高分辨率质谱检测,确定细胞裂解液可催化合成NCD。在培养基内补加5.0 mmol/L烟酰胺核糖后,工程菌胞内合成NCD达41.6 μmol/L。对工程菌进行发酵和油脂提取,发现胞内表达NCD合酶未导致细胞产油性能降低,后续可通过表达其他NCD偏好性酶,有望在圆红冬孢酵母中建立受NCD调控的油脂合成代谢体系。     

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     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.