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.     

Metabolic regulation of malic enzyme activity from Paracoccus denitrificans by glyoxylate and acetylCoA.

$\text{Paracoccus denitrificans}$ contains both NAD⁺- and NADP⁺-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH₄⁺. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP⁺-linked activity but has little effect on the NAD⁺-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell.     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

Transient Changes in the Energy State of Adenylates and the Redox States of Pyridine Nucleotides in Chlorella Cells Induced by Environmental Changes

The unicellular green algae Chlorella ellipsoidea was used tostudy transient changes in the energy state of adenylates andthe redox states of pyridine nucleotides induced by environmentalchanges. The transition from anaerobic to aerobic conditionsin the dark induced a sharp rise in the ATP ratio [ATP/(ATP+ADP+AMP)],a sudden decrease in the NADH ratio [NADH/(NAD⁺+NADPH)] anda transient drop in the NADPH ratio [NADPH/(NADP⁺+NADPH)]. Illuminationafter a dark period under anaerobic, CO₂-free conditions inducedsharp increases in the ATP and NADPH ratios and a slower decreasein the NADH ratio. Illumination under aerobic conditions, ineither the presence or absence of CO₂, caused a sharp increasein the NADPH ratio, a small increase in the ATP ratio and aslower increase in the NADH ratio. In the presence of CO₂, asubsequent large drop in the NADPH ratio occurred. Darkeningunder anaerobic, CO₂-free conditions induced a sudden decreasein the ATP ratio, a temporary fall in the NADPH ratio and aslow increase in the NADH ratio. Darkening under aerobic conditionsinduced transient drops in the ATP and NADPH ratios and a suddendrop in the NADH ratio. The addition of CO₂ to the atmospherewith illumination produced a decrease in all three parameters. These results are discussed in relation to current theoriesof the interaction between photosynthesis and respiration. Ourobservations indicate that the energy and reducing potentialsgenerated by photochemical processes are used for and controlother processes besides CO₂ fixation in photosynthetic cells. (Received December 3, 1981; Accepted May 4, 1982)     

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.     

The distribution of the NADPH regenerating mannitol cycle among fungal species

The mannitol cycle is an important NADPH regenerating system in Alternaria alternata. The cycle is built up of the following enzymes: mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, mannitol dehydrogenase and hexokinase. The net reaction of one cycle turn is: NADH+NADP⁺+ATP NAD⁺+NADPH+ADP+P_i. The enzymes needed for an operating cycle were found in Aspergillus, Botrytis, Penicillium, Pyricularia, Trichothecium, Cladosporium and Thermomyces all genera belonging to Fungi Imperfecti. The only genus of this class lacking the cycle was Candida. No genera from the classes Basidiomycetes and Phycomycetes showed any mannitol 1-phosphate dehydrogenase or mannitol 1-phosphatase activities. The genera investigated, belonging to Ascomycetes, Gibberella, Ceratocystis and Neurospora all lacked mannitol 1-phosphate dehydrogenase. It was concluded that the mannitol cycle is an important and widespread pathway for NADH oxidation and NADP⁺ reduction in the organisms belonging to the class Fungi Imperfecti.     

Pentosan polysulfate,a potent anti HIV and anti tumor agent,inhibits protein serine/threonine and tyrosine kinases

The effect of nicotinamide-adenine dinucleotides (NAD⁺ and NADP⁺) on Ca²⁺ transport in rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD⁺ (2mM) or NADP⁺ (1 and 2mM) caused a significant inhibition of Ca²⁺ uptake following addition of 2mM ATP. Ca²⁺, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD⁺ (0.5–2mM) or NADP⁺ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca²⁺ uptake and release clearly weakened in comparison with the effects of NAD⁺ and NADP⁺. Meanwhile, ryanodine (10M), thapsigargin (10M) or oxalate (0.5mM) had no effect on Ca²⁺ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD⁺ on Ca²⁺ uptake and release. Thus, NAD⁺ and NADP⁺ had a potent effect on Ca²⁺ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD⁺ (NADP⁺) is a factor in the regulation of the nuclear Ca²⁺ concentration. (Mol Cell Biochem121: 127–133, 1993)     

Endothelium and smooth muscle of pig coronary artery: Differences in metabolism

Here, we report that the smooth muscle and endothelium of the pig coronary artery differ in the profiles of energy metabolism nucleotides. ATP levels in the freshly isolated smooth muscle (1490 ± 93, all the values are in pmol/mg protein) were significantly greater than in the endothelium (418 ± 68). In contrast, endothelium contained higher levels of NADH (328 ± 21), NAD⁺ (1210 ± 28), NADPH (87 ± 2), and NADP⁺ (77 ± 4) than smooth muscle (17 ± 2, 96 ±14, 7 ± 1, and 8 ± 1, respectively). However, smooth muscle and endothelium do not differ from each other in the ratios of NADH/NAD⁺ or NADPH/NADP⁺. Cells cultured from smooth muscle and endothelium contained less ATP (93 ± 2, 141 ± 6) and had lower ratios of NADH/NAD⁺ than the freshly isolated tissues but the NADPH/NADP⁺ ratios remained similar. We conclude that (a) freshly isolated smooth muscle and endothelium differ in their profiles of the energy metabolism nucleotides, and (b) culturing the cells alters the profile.     

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.     

A new high performance liquid chromatographic technique for separation and determination of adenylic and nicotinamide nucleotides in Lactobacillus plantarum

A new technique for separation and determination of ATP, ADP, AMP, NAD⁺, NADP⁺, NADH and NADPH in Lacwbacillus plantarum has been developped. It involves acid and basic extraction of nucleotide pool, analysis by reverse-phase high performance liquid chromatography on a 5 m Spherisorb ODS-1 column and UV detection. The method offers advantages in accurate estimations of adenylic and nicotinamide nucleotides concentrations in Lactobacillus plantarum during the growth phase. Such results indicate the potential of this technique as an important research tool.     

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.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.     

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.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP⁺, NAD⁺ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP⁺ and NAD⁺. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O₂ or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD⁺ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.Abbreviations DTNB 5,5-dithiobis(2-nitrobenzoate) - EGTA Ethylenglycoltetraacetic Acid - TNB 5-thio-2-nitrobenzoate - Trx Thioredoxin - Trx(SH)2 Reduced Thioredoxin - Trx-S2 Oxidized Thioredoxin     

Thymidine production by overexpressing NAD<Superscript>+</Superscript> kinase in an <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant strain

Intracellular NADPH/NADP⁺ ratio in cells grown on various production media with different carbon and nitrogen sources had a positive correlation with the thymidine production. To improve thymidine production in a previously engineered E. coli strain, NAD⁺ kinase was overexpressed in it resulting in the NADPH/NADP⁺ ratio shifting from 0.184 to 0.267. The [NADH + NADP⁺]/[NAD⁺ + NADPH] ratio was, however, not significantly altered. In jar fermentation, 740 mg thymidine l⁻¹ was produced in parental strain, while 940 mg l⁻¹ of thymidine was produced in NAD⁺ kinase-expressing strain.     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.