NADPH Regeneration Research Articles

Incorporation of Two Oxygens from 18O2 in the Epoxyquinone from the Dihydroxyacetanilide Epoxidase Reaction: Evidence for a Dioxygenase Mechanism

Dihydroxyacetanilide, 4, is oxidized to the 5R,6S-epoxyquinone, 3, by dihydroxyacetanilide epoxidase-I (DHAE I) from Streptomyces LL-C10037, without the assistance of an organic cofactor. 13C NMR analysis revealed that in the presence of 18O2 a full equivalent of 18O is incorporated at the epoxide. However, control reactions revealed the rapid exchange of the C-4 carbonyl with H218O. By coupling the DHAE I reaction with 2-acetamido-5,6-epoxy-1,4-benzoquinone oxidoreductase (AEBQOR I) from the same organism, NADP, and an NADPH regeneration system based on glucose 6-phosphate dehydrogenase, the epoxyquinol LL-C10037α, 1, was produced with ∼20% incorporation of a second 18O atom at the C-4 alcohol. Therefore, DHAE I is a dioxygenase with an epoxidation mechanism essentially the same as has been observed for the dihydrovitamin K epoxidation occurring during the mammalian vitamin K-dependent glutamate carboxylase reaction.

Electromicrobial regeneration of pyridine nucleotides and other preparative redox transformations with Clostridium thermoaceticum

Clostridium thermoaceticum contains interesting enzymes suitable for redox reactions. Various AMAPOR (artificial-mediator-accepting pyridine-nucleotide oxidoreductase) activities were used for electromicrobial pyridine nucleotide regeneration. The combination of AMAPOR with commercially available pyridine-nucleotide-dependent oxidoreductases led to (S)-glutamate, (2R,3S)-isocitrate, (2S,3R)-isocitrate, 6-phosphogluconate and ribulose 5-phosphate. The redox equivalents were provided by electrochemically regenerated artificial mediators. Methylviologen or cobalt sepulchrate were used for NAD(P)H regeneration, whereas carboxamidomethylviologen (CAV) or anthraquinone sulphonates (AQ-S) were suitable for NAD(P)+ regeneration. With resting cells of C. thermoaceticum productivity numbers {mmol product/[biocatalyst (kg dry weight) × time (h)]} of about 30 000 for NADPH, 7000 for NADH and 14 000 for NADP+ regeneration could be reached. The cycle number for NADPH regeneration was up to 4300, that for NADP+ regeneration was at least 1600. An aldehyde and an alcohol oxidoreductase were used to reduce non-activated carboxylic acids to the alcohols and to dehydrogenate primary alcohols to the aldehydes or carboxylates. The electromicrobial reduction of 6-chloropyridine 3-carboxylate to the corresponding alcohol was compared with the reduction by CO as electron donor. The application of phenothiazine-dye-type mediators (thionine, methylene blue) converted primary alcohols to the aldehydes with productivity numbers up to 1400 in the presence of hydrazine as aldehyde scavenger. With CAV or AQ-S, alcohols were dehydrogenated to carboxylic acids with productivity numbers of almost 1700.

Redox reaction system conjugating electrochemical reduction of NADP + and enzymatic reaction across the electron transfer membrane

The conjugated redox reaction was driven across the electron transfer membrane prepared from a urethane prepolymer to carry positive charge, where NADP + as electron transfer carrier was adsorbed in the prepared polyurethane membrane. Glutathione reductase [NAD (P)H: oxidized-glutathione oxidoreductase (EC 1.6.4.1)] was used as the catalyst for production of the reduced form of glutathione (GSH) from the oxidized form (GSSG) in the objective reaction, and methyl viologen (MV) was used for the electrochemical regeneration of NADPH in the subreaction. The conjugated redox reaction in the separated reactions system, using the three-compartment cell with two membranes, was successful without MV contamination. Under the given conditions, the conversion ratio of GSH from GSSG reached 50% after 4 h of incubation at 30°C and the amount of GSH accumulated was 0.5 μmol ml −1 of reaction mixture.

Kinetic mechanism of the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase

The reaction of oxidation of 2-propanol to acetone in the presence of NADP catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH) has been studied for gaining insight into the coupled-substrate approach that has been extensively used for NADPH regeneration in preparative-scale reductions catalyzed by this enzyme. The data obtained in initial velocity experiments in the absence of products as well as those involving product inhibition and dead-end inhibition by pyrazole suggest that the reaction mechanism is of the Theorell-Chance BiBi type and in which the coenzymes bind to the free form of the enzyme. The seven kinetic parameters that characterize this kinetic model were estimated by non-linear regression analysis using the appropriate rate equations. The kinetic mechanism proposed in the present work is fully compatible with the coupled-substrate approach used for regeneration of NADPH in reduction reactions catalyzed by TBADH.

NAD(P)H regeneration using ethanol as an energy source in baker's yeast-mediated bioreduction

A scheme for NAD(P)H regeneration from the corresponding oxidized form through the oxidative pathway of ethanol in baker's yeast cells was studied. Baker's yeast-mediated bioreduction of carbonyl compounds coupled with the oxidative pathway of ethanol is efficient and clean compared with the earlier procedure using carbohydrates as the energy source. To study NADH regeneration, the bioreduction of acetol catalyzed by NADH-dependent carbonyl-reducing enzymes was examined by using ethanol or acetate as the energy source under several aeration conditions, including an anaerobic environment. The results suggested that NAD + formed on the reduction of the carbonyl group was mainly regenerated as NADH through alcoholic oxidation into acetate. NADPH regeneration was then studied in the same manner by examining the bioreduction of ethyl acetoacetate catalyzed by NADPH-dependent carbonyl-reducing enzymes. The results suggested that NADPH was regenerated through acetate oxidation into carbon dioxide. Thus, a total scheme for NAD(P)H regeneration using ethanol as the energy source, coupled with bioreduction, was proposed.

Characteristics of the Mannitol Pathway in a Root Hemiparasitic Species, Thesium humile Vahl. (Santalaceae)

The root hemiparasite, Thesium humile Vahl., causes damage in cereal crops in Mediterranean countries. It is studied here growing on wheat, from which it differs by the main accumulated soluble carbohydrate. Thesium contains effectively large amounts of mannitol while sucrose is typically accumulated in the host. The mannitol pathway may be thus a metabolic target for a specific chemical control of this hemiparasite. Identification and characterization of intermediary metabolites and enzymes required for mannitol production are related in this paper. Mannitol biosynthesis from CO2 fixation occurred in aerial parts and especially in the young leaves. Fructose-6-P (F6P), resulting from the triose-phosphate (TP) conversion, is at first converted to mannose-6-P (M6P) with the mannose-6-P isomerase (M6PI). With the NADP-dependent mannose-6-P: mannitol-1-P oxydoreductase (M6PR), M6P is then reduced to mannitol-1-P (M-1P), which is finally dephosphorylated with the mannitol-1-phosphate phosphatase (M1Pase). The values of optimum pH of these enzymes agree with a cytosolic location of the mannitol biosynthesis. Mannitol production is greatly regulated by temperature, notably concerning the M-1Pase. No correlation between mannitol export and increased mannitol synthesis has been observed in the expanding leaves. Activities are compared with those measured in other mannitol-synthesizing parasitic genera, such as Orobanche and Osyris. M6PI, and M-1Pase then appear as the limiting enzymes of the mannitol pathway in these plants. The role of the phosphate-pentose pathway (PPP) in the regeneration of the cytosolic NADPH required for mannitol biosynthesis is also discussed. These results allow to envisage further assays to inhibit the mannitol pathway in Thesium leaves, in order to develop a specific chemical control of this hemiparasitic weed in the future.

Yield of poly‐D(‐)‐3‐hydroxybutyrate from various carbon sources: A theoretical study

The theoretical yield of poly-D(-)-3-hydroxybutyrate (PHB) has been estimated from the biochemical pathway leading to PHB when a carbohydrate (glucose), a C(1) compound (methanol), a C(2) compound (acetic acid), or a C(4) compound (butyric acid) is used as a carbon source. In estimating the yield, recycling (or regeneration) of NADP(+)/ (NADPH + H(+)) and NAD(+) /(NADH + H(+)) have been taken into account. A special emphasis is made on te regeneration of NADPH, which is the coenzyme of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADPH-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. An equation which predicts the overall yield of PHB when non-PHB residual biomass is actually formed has been derived as a function of both the theoretical yield and PHB content of the dry cell mass. The ratio of the overall yield to the theoretical yield is roughly proportional to the PHB content.

Effects of the Phenacetin Metabolite 4-Nitrosophenetol on Glycolysis and Pentose Phosphate Pathway in Human Red Cells

Human erythrocytes exposed to 4-nitrosophenetol showed marked alterations of their endogenous metabolism. Rapid ferrihemoglobin formation mediated by the NADPH-dependent enzymic cycling of the nitrosoarene ("Kiese cycle") and extensive GSSG production caused an immediate drain of G-6-P into the pentose phosphate pathway at maximal flow. Despite a 2.4-fold increase in glucose phosphorylation rate and a branching ratio of 97:3 between pentose phosphate pathway and Embden-Meyerhof pathway, the G-6-P supply was obviously insufficient to meet the immense NADPH demand. Thus, a significant recycling of pentose phosphate pathway-derived F-6-P was observed in the order of 65%. Comparison of NADPH regeneration and ferrihemoglobin formation indicates the "Kiese cycle" to be a minor mechanism in ferrihemoglobin production in the case of high 4-nitrosophenetol concentrations. Most probably, reactive intermediates of 4-nitrosophenetol other than N-hydroxy-4-phenetidine, i.e. bicyclic arylamines and glutathione S-conjugates are formed which produce ferrihemoglobin without involvement of NADPH. The experiments have shown that red cells are remarkable robust to tackle the massive oxidative stress as elicited by 4-nitrosophenetol. The immediate metabolic response of the pentose phosphate pathway allows rapid regeneration of reduced glutathione. Thereby, SH-containing enzymes are effectively protected and/or regenerated and hemolysis is kept minimal. Hence, red cells are favourably suited for clearing the blood from N-oxygenated arylamines before they can reach more sensitive target organs.

Lactobacillus kefir alcohol dehydrogenase: a useful catalyst for synthesis

The alcohol dehydrogenase from Lactobacillus kefir simultaneously catalyzes carbonyl reductions and NADPH regeneration in the presence of 2-propanol. Representative synthesis of a number of chiral alcohols was accomplished in good yield and high enantiomeric excess (94-99%). This NADPH-requiring enzyme tranfers the pro-R hydride from the cofactor to the si face of carbonyls to give (r) alcohols

Photochemical regeneration of NADPH using the enzyme ferredoxin-NADP + reductase

Ferredoxin-NADP + reductase and the electron transfer protein ferredoxin (or flavodoxin) were used to photochemically regenerate NADPH. The presence of ferredoxin enhanced the rate of NADPH production almost fivefold when either proflavin or 5-deazariboflavin was used as photosensitizer. The very high turnover numbers (8,100 min −1) obtained for the enzyme in this system indicate the efficiency of the process. The system was also shown to be very stable, since total turnover numbers of approximately 3.2 × 10 5 were obtained during continuous operation.

NADPH regeneration by glucose dehydrogenase from Gluconobacter scleroides for l-leucovorin synthesis.

A new process for (6S)-tetrahydrofolate production from dihydrofolate was designed that used dihydrofolate reductase and an NADPH regeneration system. Glucose dehydrogenase from Gluconobacter scleroides KY3613 was used for recycling of the cofactor. The reaction mixture contained 200 mM dihydrofolate, 220 mM glucose, 2 mM NADP, 14.4 U/ml dihydrofolate reductase, and 14.4 U/ml Glucose dehydrogenase, and the reaction was complete after incubation at pH 8.0, and 40 degrees C for 2.5 hr. With (6S)-tetrahydrofolate as the starting material, l-leucovorin was synthesized via a methenyl derivative. The purity of the l-leucovorin was 100%, and its diastereomeric purity was greater than 99.5% d.e. as the (6S)-form.

Enzymes Involved in the NADPH Regeneration System Coupled with Asymmetric Reduction of Carbonyl Compounds in Microorganisms.

Enzymes Involved in the NADPH Regeneration System Coupled with Asymmetric Reduction of Carbonyl Compounds in Microorganisms.

Increase in eukaryotic initiation factor 2B activity following fertilization reflects changes in redox potential.

One of the factors involved in the postfertilization activation of protein synthesis in the sea urchin, Strongylocentrotus purpuratus, is the activation of eIF-2B, the initiation factor responsible for guanine nucleotide exchange on eIF-2. Cell-free translation systems from unfertilized eggs are stimulated by added eIF-2B, although this dependency is rapidly lost in translation systems prepared at various times following fertilization. Cell-free translation systems prepared from unfertilized eggs show significantly lower eIF-2B activities than those prepared from 2-h embryos. However, the provision of an NADPH regeneration system significantly stimulates eIF-2B activity in egg extracts and, in addition, stimulates both binding of initiator tRNA to the small ribosomal subunit and protein synthetic activity. These data suggest that the activation of eIF-2B following fertilization reflects the fertilization-induced increase in NADPH levels.

Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir

A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R(+)-phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg2+ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on NADPH and was inactive when supplied with NADH as the coenzyme. Important enzymological data of the dehydrogenase are: Km (acetophenone) 0.6 mM, Km (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of NADPH: glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.

Photochemically Induced Oxidative and Reductive Regeneration of NAD(P)+/NAD(P)H Cofactors: Applications in Biotransformations

AbstractPhotosensitized regeneration of NAD(P)H cofactors is accomplished by biocatalyzed and artificially catalyzed transformations in photochemical assemblies. Photogenerated N,N'‐dimethyl‐4,4′‐bipyridinium radical cation, MV+., acts as electron carrier for the reduction of NADPH in the presence of the enzyme ferredoxin reductase and for the reduction of NADH in the presence of lipoamide dehydrogenase. For the photogeneration of MV+. and subsequent NADPH formation, three different photosensitizers are applied: Ru(bpz)23+, Ru(bpy)23+, and Zn—TMPyP4+. The highest quantum yield for NADPH formation is observed with Ru(bpz)32+ and is ϕ = 1.7 × 10−1. For NADH regeneration only Zn‐TMPyP4+ can be applied. Ru(bpy)32+ and Ru(bpz)2+3 interact with NADH in their excited or oxidized forms and therefore cannot be used as light‐active compounds in the system. The NADPH regeneration cycle has been coupled to the biocatalyzed synthesis of glutamic acid. Although Ru(bpz)32+ is 42.5‐fold more efficient than Ru(bpy)32+ in the regeneration of NADPH, the synthesis of glutamic acid is improved only by a factor of 2 in the presence of Ru(bpz)32+, implying that the coupled process is rate limiting. Oxidative regeneration of the NAD+ cofactor is accomplished in a photosystem that includes Ru(bpy)32+ as photosensitizer. The photoprocess is coupled to dehydrogenation of ethanol, propanol, lactic acid, and alanine with concomitant H2 evolution. A photosystem that includes Ru(bpy)32+ as photosensitizer, ascorbate as electron donor, and chloro‐tris‐(3‐diphenylphosphinobenzene sulfonate)Rh(I), RhCl(dpm)33‐, is catalytically active in the photoinduced regeneration of NAD(P)H cofactors. Mechanistic investigations show that photogenerated Ru(bpy)3+ mediates the generation of a hydrido‐rhodium complex that acts as a charge relay for the production of NAD(P)H.

Regeneration of NADPH and Hydrogenation of Ketones to Alcohols with the Combination of Hydrogenase, Ferredoxin-NADP Reductase, and Alcohol Dehydrogenase

Abstract The regeneration of nicotinamide–adenine dinucleotide phosphate (reduced form, NADPH) by the reduction of NADP by hydrogen gas was carried out with the [hydrogenase–ferredoxin-NADP reductase (FNR)] system. Ketone hydrogenation was tried by the combination of the above system and alcohol dehydrogenase (ALDH), as shown in the following scheme: (Remark: Graphics omitted.) In the [hydrogenase–FNR] system, NADP was easily reduced by hydrogen to form NADPH. By adding ALDH to the above system, the hydrogenation of ketone to alcohol was observed. The ketones used in this study were 2-butanone, 2-pentanone, and 2-hexanone. After 8 h, the turnover number of NADP was 13 for 2-butanone, 15 for 2-pentanone, and 10 for 2-hexanone, showing a recurrence of NADP by hydrogen gas.

On a viologen-dependent pyridine nucleotide oxidoreductase from a thermophilic Bacillus

From a thermophilic bacillus a viologen dependent pyridine nucleotide oxidoreductase has been purified and partially characterized. Its apparent molecular weight is about 120000 consisting of two subunits of equal or very similar molecular weight. Per molecular weight of 120000 the enzyme contains 4 FAD. FMN or labile sulfur could not be detected. The physiological role of the enzyme is not clear. It reduces NAD as well as NADP at the expense of reduced methylviologen. The reduced pyridine nucleotides can be reoxidized with carbamoylmethylviologen. The seven determined Km- and six Ki-values show that the enzyme is suitable for the regeneration of NADH, NADPH, NAD and NADP. The stability in presence of oxidized and reduced methylviologen at 35°C or 60°C is satisfying for preparative work.

Tert.-Butyl hydroperoxide metabolism and stimulation of the pentose phosphate pathway in isolated rat hepatocytes

The metabolism of tert.-butyl hydroperoxide (TBHP) by the glutathione peroxidase/reductase system in isolated hepatocytes results in the rapid depletion of reduced glutathione and NADPH. The regeneration of NADPH can occur through the pentose phosphate pathway, but only when the pathway is stimulated, for example, by NADP + and possibly oxidized glutathione, both of which can be elevated in hepatocytes exposed to TBHP. TBHP is a cytotoxicant and the role of NADPH and the pentose phosphate pathway in protecting hepatocytes from TBHP-induced injury is unknown. Isolated rat hepatocytes exposed to TBHP (0.5 m m) for 30 min metabolized more [1- 14C]glucose to 14CO 2 than control (638.2 ± 96.2 vs 306.9 ± 69.5 dpm/10 6 cells) whereas 14CO 2 evolution from [6- 14C]glucose was unchanged, indicating that TBHP increases the activity of the pentose phosphate pathway and not glycolysis. TBHP (0.25 m m) metabolism also resulted in a rapid oxidation of hepatocyte NADPH from 2.85 ± 0.32 to 0.55 ± 0.24 nmol/10 6 cells which rapidly returned to 3.58 ± 0.27 nmol NADPH/10 6 cells. Inhibition of the pentose phosphate pathway with 6-aminonicotinamide (70 mg/kg; 5 hr prior to hepatocyte isolation) inhibited TBHP-stimulated 14CO 2 evolution from [1- 14C]glucose and decreased the rate of NADP + reduction. Hepatocytes isolated from 6-aminonicotinamide-treated animals were more susceptible to TBHP-induced cell injury than were control hepatocytes. These data demonstrate the following: (1) The metabolism of TBHP by isolated hepatocytes stimulated the activity of the pentose phosphate pathway; and (2) inhibition of the pentose phosphate pathway with 6-aminonicotinamide potentiated the toxicity of TBHP to isolated rat hepatocytes. These results suggest that the regeneration of NADPH by the pentose phosphate pathway may play a significant role in protecting hepatocytes from TBHP-induced damage.

Photoinduced enzyme-catalysed synthesis of amino acids by visible light

Visible light-induced NADPH regeneration effects the production of glutamic acid that mediates transamination and formation of aspartic acid and alanine in the presence of enzymes.