Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH,NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8–9 U mg^?1 protein (µmoles mg^?1 min^?1) for NADPH and 3–4 U mg^?1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^?1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^?1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^?1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

The NAD⁺‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD⁺ and exhibits only a low activity with NADP⁺ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD⁺. While an application in the regeneration of NADP⁺ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP⁺‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP⁺ acceptance. The mutant V39R was identified to have increased activity with NADP⁺ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP⁺ in an enzyme‐coupled approach combined with the NADP⁺‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP⁺.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

Partial Purification and Characterization of NADP-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.)

NADP⁺-isocitrate dehydrogenase (threo-DS-isocitrate: NADP⁺ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg²⁺ or Mn²⁺, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP⁺, dl-isocitrate, Mn²⁺, and Mg²⁺. Their K_m values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP⁺. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP⁺ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP⁺ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP⁺-IDH in situ is likely to be controlled by intracellular NADPH to NADP⁺ ratio as well as by the concentration of various substrates and products.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Regeneration of the nicotinamide cofactor using a mediator-free electrochemical method with a tin oxide electrode

Tin (IV) oxide was made using an anodization and annealing method and was used as a working electrode in an electrochemical cofactor regeneration reaction. This material was formed with a large surface area, and by changing the preparation conditions, it was possible to control the morphology. Tin oxide has redox properties similar to those of frequently used mediators required for electron transfer between cofactors and an electrode. Therefore, by using tin oxide as a novel electrode, mediator-free electrochemical cofactor regeneration may be possible. Oxidation and reduction of the nicotinamide cofactors, NAD(P)H and NAD(P)⁺, were carried out under various reaction conditions. The results showed a high efficiency for oxidizing NADH over a broad range of pH and temperatures. The oxidation tendency of NADPH was also observed, and it demonstrated a similar reaction tendency as NADH. When using a tin oxide electrode, NAD⁺ was readily reduced to NADH, though the efficiency of this reaction was lower than for NADH oxidation. Oxidation of 2-propanol to acetone was used as a model system using alcohol dehydrogenase and the cofactor regeneration system suggested in this study. The electroenzymatic reaction showed efficient regeneration of NADP⁺ without a mediator.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Enzymatic production of ethyl (R )-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP⁺, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP⁺, based on the amounts of NADP⁺ added and CHBE formed, was about 5100 mol/mol. Received: 26 May 1997 / Received revision: 16 July 1997 / Accepted: 29 August 1997     

Der Einfluß der Kulturbedingungen auf den Nicotinamid-Adenin-Dinucleotid(phosphat)-Gehalt in Zellen von Rhodospirillum rubrum

Zusammenfassung In Zellen von R. rubrum war das Verhältnis von oxydiertem zu reduziertem NAD(P) vom Sauerstoffpartialdruck im Medium, der Lichtintensität und der Nährbodenzusammensetzung abhängig. In ruhenden Kulturen unter aeroben Bedingungen im Licht oder im Dunkeln und anaerob bei hoher Lichtintensität, wenn der ATP-Pool in den Zellen groß ist, beobachtete man einen relativ hohen Wert für das Verhältnis von NAD(P)⁺/NAD(P)H. Unter Kulturbedingungen, bei denen der ATP-Gewinn der Zellen gering ist (anaerob Schwachlicht oder anaerob Dunkel), sank das Verhältnis von NAD(P)⁺/NAD(P)H ab. Die niedrigsten Werte für das Verhältnis von NAD(P)⁺/NAD(P)H wurden dementsprechend in anaerober Dunkelkultur, die höchsten in aerober Lichtkultur gefunden.Anaerob im Dunkeln war der NAD(P)H-Spiegel auch vom Substrat abhängig: mit Fructose oder ohne Substrat beobachtete man einen sehr großen NAD(P)H-Pool in den Zellen; nach Zugabe von Acetat, Succinat, Pyruvat oder Malat sank der Spiegel der reduzierten Coenzyme ab.In wachsenden Kulturen (außer anaerob im Dunkeln) nahm die relative Konzentration von NAD⁺ und der NADP⁺-Pool im Vergleich zu ruhenden Zellen stark zu (3-5fach).Änderungen im Verhältnis von NAD⁺/NADH und von NADP⁺/NADPH waren aber nicht unter allen Kulturbedingungen direkt korreliert.Es wird diskutiert, wieweit das Adenylatsystem und das NAD(P)-System einen regulativen Einfluß auf die Bacteriochlorophyll-Synthese und die Morphogenese bei Athiorhodaceae haben.

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The influence of culture conditions on the NAD(P) content of Rhodospirillum rubrum cells

Summary In cells of R. rubrum the ratio of oxidized to reduced NAD(P) depended on the oxygen pressure in the medium, the light intensity, and the composition of the medium. The ratio of NAD(P)⁺/NAD(P)H was high under conditions when the ATP-pool in the cell is large, viz. in resting cultures either kept aerobically in the light or in the dark or kept anaerobically in strong light. The quotient NAD(P)⁺/NAD(P)H decreased under conditions of reduced ATP-synthesis in the cells (anaerobic in dimlight or in the dark). Consequently, the lowest NAD(P)⁺/NAD(P)H value was observed in anaerobic dark cultures, the highest in aerobic light cultures.Under anaerobic conditions in the dark, the NAD(P)H level depended also on the substrate: with fructose or without any substrate, a large NAD(P)H pool was observed; the level of reduced coenzymes decreased upon addition of acetate, succinate, pyruvate, or malate.In growing cultures (except under anaerobic conditions in the dark) the relative concentration of NAD⁺ and the NADP⁺ pool showed a considerable increase (3 to 5 fold), as compared with resting cells. However, the changes in the proportions of NAD⁺/NADH and NADP⁺/NADPH were not directly correlated under all culture conditions.The regulative influence of the adenylate and the NAD(P) systems on the synthesis of bacteriochlorophyll and morphogenesis in Athiorhodaceae is discussed.

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Abkürzungen BChl Bacteriochlorophyll a - NAD(P) NAD-Nucleotide=reduziertes und oxydiertes Nicotinamid-Adenin-Dinucleotid und Nicotinamid-Adenin-Dinucleotidphosphat Herrn Prof. Dr. H. Engel zum 70. Geburtstag gewidmet.     

Metabolisme du γ-aminobutyrate chez Agaricus bisporus III. La succinate-semialdehyde:NAD(P)+ oxydoreductase

Metabolism of γ-Aminobutyrate in Agaricus bisporus. III. The Succinate-Semialdehyde: NAD (P)⁺ Oxidoreductase. The succinate-semialdehyde:NAD(P)⁺ oxidoreductase (E.C. 1.2.1.16) is responsible for the second step in the catabolism of γ-aminobutyrate: the irreversible enzymatic conversion of succinic semialdehyde (SSA) to succinate. Succinate semialdehyde dehydrogenase was extracted from mitochondrial fraction of fruit-bodies of Agaricus bisporus Lge. The mitochondrial pellet was sonicated and centrifuged at 110,000 g; the supernatant obtained was designated the “crude extract”. The enzyme was extremely unstable on storage, unless 1 mM EDTA and 20% glycerol were added. Kinetic studies were carried out at 30°C, and the formation of NADH or NADPH was followed by measuring increase of absorbance at 340 nm with a spectrophotometer. The dehydrogenase was completely inactive when the reaction was run in the absence of thiol and was more active with NAD⁺ than with NADP⁺. In the “crude extract” the activity with NADP⁺ had a pH optimum between 8.6 and 9.1 and the K_m values for SSA and NADP⁺ were 2.0 × 10^?4M and 1.4 × 10^?4M respectively. The pH optimum with NAD⁺ was found between 8.6 and 8.8 and the K_m value for SSA is 4.8 × 10^?4M and for NAD⁺ 2.0 × 10^?3M. With NAD⁺, the kinetic values (pH, K_m) of the “crude extract” chromatographed on hydroxylapatite were unchanged. Inhibition by thiamine pyrophosphate (TPP) was uncompetitive with respect to NAD⁺, those by malate, ATP, ADP and NADPH non-competitive and that by NADH competitive. These results and the fact that activity with NAD⁺ was lost more slowly than with NADP⁺ indicate the possibility of at least two mitochondrial succinate-semialdehyde dehydrogenases, even though the activities of this enzyme assayed with NAD⁺ and NADP⁺ respectively were not able to be separated from each other by hydroxylapatite column chromatography. Some speculations on the metabolic regulation of this dehydrogenase and considerations on the significance of these results in the physiology of respiration in Agaricus bisporus Lge are given.     

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP⁺, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP⁺ and NAD⁺. One of the XDH variants utilized NADP⁺ about 4 times more efficiently than NAD⁺. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.     

Cytosolic NADPH is the electron donor for extracellular fe reduction in iron-deficient bean roots

Pyridine nucleotides were determined in lateral roots of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prelude. In iron-deficient plants, total NADP per gram fresh weight and the NADPH/NADP⁺ ratio were twice the values found in iron-sufficient plants. The NADPH/NADP⁺ ratio in iron-deficient plants was considerably lowered after a 2 minute incubation in 1 millimolar ferricyanide. Total NAD was not influenced by growth conditions and was mainly present in oxidized form.

These results indicate that NADPH is the electron donor for the high Fe^III reduction activity found in iron-deficient roots, a process that is part of the Fe-uptake mechanism.

     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

Isocitrate dehydrogenase from Rhodopseudomonas spheroides: kinetic mechanism and further characterization

Product inhibition studies with Rhodopseudomonas spheriodes NADP⁺ specific isocitrate dehydrogenase indicate that the enzyme mechanism involves the ordered addition of the substrates NADP⁺ and threo-d_s-isocitrate and the ordered release of products CO₂ (HCO_s^?), 2-ketoglutarate, and NADPH. In addition, the presence of a ternary complex consisting of enzyme, NADP⁺, and 2-ketoglutarate is indicated. Binding studies with radioactive substrates support the kinetically derived mechanism. The Rhodopseudomonas enzyme is dimeric and contains but a single active site. Different combinations of substrate were ineffective in causing gross changes in molecular structure as monitored by gel filtration techniques. A comparison of the amino acid composition of this enzyme with the bacterial enzyme from Azotobacter vinelandii indicate very significant differences in the amino acid compositions.     

Reactivation of Inactivated d-Glucose Dehydrogenase of a Bacillus Species by Pyridine and Adenine Nucleotides

d-Glucose dehydrogenase [β-d-glucosc: NAD(P) oxidoreductase (EC 1.1.1.47)] was synthesized derepressively in a mutant of a Bacillus species which was isolated as an improved strain for d-ribose production. The enzyme was very unstable and inactivated during storage or column chromatography. The inactivation was prevented in the presence of NAD⁺, NADP⁺ or certain salts. The inactive enzyme was reactivated by the addition of NAD⁺, NADH, NADP⁺, NADPH, AMP, ADP, ATP or certain salts. The molecular weights of the inactive and active form of the enzyme were estimated to be about 45,000 and 80,000, respectively, by Sephadex G–150 gel filtration. Thus, it seems that the enzyme activity is regulated by monomer-dimer interconversion of the enzyme molecule.     

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia inParacoccus denitrificans. Glutamate dehydrogenase (NADP⁺) catalyzes the assimilation at a high NH₄ ⁺ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH₄ ⁺ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP⁺) and glutamate-ammonia ligase inP. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP⁺). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP⁺) by the high concentration of glutamine or its metabolic products as in the case when NH₄ ⁺ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP⁺) was observed.