The Isolation,Purification, and Some Properties of NAD-Dependent Isocitrate Dehydrogenase from the Organic Acid–Producing Yeast Yarrowia lipolytica

The NAD⁺-dependent isocitrate dehydrogenase of the organic acid–producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The K _m for NAD⁺ is 136 M, and that for isocitrate is 581 M. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD⁺) in the overproduction of citric and keto acids is discussed.     

Activity of liver mitochondrial Krebs cycle NAD<Superscript>+</Superscript>-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD⁺/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD⁺/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD⁺-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD⁺/NADН ratio.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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 new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

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.     

Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation

Malic enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO₂ providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD⁺ as the primary cofactor. K_m values for NAD⁺ and NADP⁺ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP⁺- but not NAD⁺-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP⁺-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

Overexpression of the <Emphasis Type="Italic">ICL1</Emphasis> gene changes the product ratio of citric acid production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric (CA) and isocitric (ICA) acids, triggered by growth limitation caused by different factors and an excess of carbon source. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether the CA/ICA product ratio can be influenced by gene-dose-dependent overexpression or by disruption of the isocitrate lyase (ICL)-encoding gene ICL1, recombinant Y. lipolytica strains were constructed, which harbour multiple ICL1 copies or a defective icl1 allele. The high-level expression of ICL in ICL1 multicopy integrative transformants resulted in a strong shift of the CA/ICA ratio into direction of CA. On glycerol, glucose and sucrose, the ICA proportion decreased from 10–12% to 3–6%, on sunflower oil or hexadecane even from 37–45% to 4–7% without influencing the total amount of acids (CA and ICA) produced. In contrast, the loss of ICL activity in icl1-defective strains resulted in a moderate 2–5% increase in the ICA proportion compared to ICL wild-type strains on glucose or glycerol.     

Expression of NAD<Superscript>+</Superscript>-dependent formate dehydrogenase in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> and its involvement in anaerobic metabolism and H<Subscript>2</Subscript> production

An expression system for NAD⁺-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H₂ yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H₂ production.     

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids

The yeast Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.     

New recombinant strains of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil

The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3: 1) as compared to the parental strain (1.1: 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5: 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.     

The effect of<Emphasis Type="Italic"> pfl</Emphasis> gene knockout on the metabolism for optically pure <Emphasis Type="SmallCaps">d</Emphasis>-lactate production by<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The effect of gene knockout on metabolism in the pflA^–, pflB^–, pflC^–, and pflD^– mutants of Escherichia coli was investigated. Batch cultivations of the pfl ^– mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA^– and pflB^– mutants, but not pflC^– and pflD^– mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD⁺ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA^– and pflB^– mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD⁺ ratio in pfl ^– mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.     

Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> MA-4680

A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP⁺ as electron acceptor. Apparent K _m values were 4.98 μM for NADP⁺ and 6,620 μM for NAD⁺, respectively, using Mn²⁺ as divalent cation. The enzyme performed a 33,000-fold greater specificity (k _cat/K _m) for NADP⁺ than NAD⁺. Moreover, SaIDH activity was entirely dependent on the presence of Mn²⁺ or Mg²⁺, but was strongly inhibited by Ca²⁺ and Zn²⁺. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP⁺.     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

Metabolism of Yarrowia lipolytica Grown on Ethanol under Conditions Promoting the Production of α-Ketoglutaric and Citric Acids: A Comparative Study of the Central Metabolism Enzymes

A comparative study of the enzymes of tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing -ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD<Superscript>+</Superscript>-Reducing Hydrogenase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> H16

Inclusion of an oligomeric enzyme, NAD⁺-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD⁺/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 782–789.Original Russian Text Copyright © 2005 by Tikhonova, Kurkin, Klyachko, Popov.     

Characterization of NADP<Superscript>+</Superscript>-specific <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose dehydrogenase from the thermoacidophilic Archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Thermoplasma acidophilum utilizes l-rhamnose as a sole carbon source. To determine the metabolic pathway of l-rhamnose in Archaea, we identified and characterized l-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of l-rhamnose to l-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized l-rhamnose, l-lyxose, and l-mannose using only NADP⁺ as a cofactor, which is different from NAD⁺/NADP⁺-specific bacterial RhaDs and NAD⁺-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward l-rhamnose at 60 °C and pH 7. The K _m and k _cat values were 0.46 mM, 1,341.3 min⁻¹ for l-rhamnose and 0.1 mM, 1,027.2 min⁻¹ for NADP⁺, respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP⁺-specific RhaD.