Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Action of sulphite on the substrate kinetics of chloroplastic NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two K_m values. Activation by NADPH + DTT increases V_max of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even V_max is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher V_max. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively.     

Modification of substrate specificity in single point mutants of Agrobacterium tumefaciens type II NADH dehydrogenase

Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.     

Yeast glutathione reductase. Steady-state kinetic studies of its transhydrogenase activity.

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH (K_i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate (K_i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.     

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase.

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO₄ and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M_r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M_r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 mm), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the pK of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

The effect of o-diphenols upon the microsomal NADPH and NADH oxidase activities

Catechol and catecholamines have been assayed upon the microsomal NADPH and NADH oxidase activities. Epinephrine shows a catalytic effect on the NADPH oxidation characterized by a small lag. The two to threefold increase in rate can be suppressed by Superoxide dismutase if the enzyme is added before the reaction begins. The catalytic effect is ascribed to a quinone formed by two electron oxidation of epinephrine by the Superoxide ion. The quinone, which is not catalytically active in the NADH chain, appears to mediate electrons between the NADPH-cytochrome c reductase and oxygen. The four electron oxidation product adrenochrome is also active upon the NADPH chain but inactive upon the NADH chain.Epinephrine did not change the menadione-stimulated NADPH oxidase activity. Presumably, during this and the NADH oxidase activities, two electrons are simultaneously transferred to the oxygen molecule.Catechol and catecholamines doubled the rate of autoxidation of NADH in the presence of catalytic amounts of NADH-cytochrome b₅ reductase and cytochrome b₅, a result which suggests Superoxide ion formation in the autoxidation of the cytochrome.Epinephrine does not act upon the desaturation of endogenous substrate or upon endogenous lipid peroxidation.     

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K_m^app value for d-xylose was 8.6 mM and the V_max^app was 20.8 units/mg NADH was used as a coenzyme. The K_m^app values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.     

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH₂PO₄ and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).     

The enzymic reduction of glyoxylate and hydroxypyruvate in leaves of higher plants

Glyoxylate and hydroxypyruvate are metabolites involved in the pathway of carbon in photorespiration. The chief glyoxylate-reducing enzyme in leaves is now known to be a cytosolic glyoxylate reductase that uses NADPH as the preferred cofactor but can also use NADH. Glyoxylate reductase has been isolated from spinach leaves, purified to homogeneity, and characterized kinetically and structurally. Chloroplasts contain lower levels of glyoxylate reductase activity supported by both NADPH and NADH, but it is not yet known whether a single chloroplastic enzyme catalyzes glyoxylate reduction with both cofactors. The major hydroxypyruvate reductase activity of leaves has long been known to be a highly active enzyme located in peroxisomes; it uses NADH as the preferred cofactor. To a lesser extent, NADPH can also be used by the peroxisomal enzyme. A second hydroxypyruvate reductase enzyme is located in the cytosol; it preferentially uses NADPH but can also use NADH as cofactor. In a barley mutant deficient in peroxisomal hydroxypyruvate reductase, the NADPH-preferring cytosolic form of the enzyme permits sufficient rates of hydroxypyruvate reduction to support continued substrate flow through the terminal stages of the photosynthetic carbon oxidation (glycolate/glycerate) pathway. The properties and metabolic significance of the cytosolic and organelle-localized glyoxylate and hydroxypyruvate reductase enzymes are discussed.     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.     

An extracellular NADH-oxidizing peroxidase produced by a lignin-degrading basidiomycete,Phanerochaete chrysosporium

A lignin-degrading basidiomycete, Phanerochaete chrysosporium, produces an extracellular peroxidase which in turn produces H₂O₂ by catalyzing the oxidation of NADH and NADPH. The high enzyme activity was observed in the culture grown under nutrient nitrogen limitation (low-N) and high oxygen tension (high-O₂). The enzyme activity was absent in non-ligninolytic agitated culture and in the cultures of non-ligninolytic mutant strains of this organism. The culture method using polyurethane foam cubes as a support for the growing mycelia showed the beneficial effect of producing a large amount of the enzyme. The enzyme is capable of catalyzing the oxidation of NADH and NADPH in the absence of added H₂O₂, and its activity was inhibited strongly by catalase and superoxide dismutase. It is suggested that this peroxidase participates in the ligninolytic system of Phanerochaete chrysosporium as a donor of H₂O₂, which is required for the lignin-peroxidase reaction, by oxidizing extracellular NADH and NADPH.     

NADH- and NAD(P)H-Nitrate Reductases in Rice Seedlings

By use of affinity chromatography on blue dextran-Sepharose, two nitrate reductases from rice (Oryza sativa L.) seedlings, specifically, NADH:nitrate oxidoreductase (EC 1.6.6.1) and NAD(P)-H:nitrate oxidoreductase (EC 1.6.6.2), have been partially separated. Nitrate-induced seedlings contained more NADH-nitrate reductase than NAD(P)H-nitrate reductase, whereas chloramphenicol-induced seedlings contained primarily NAD(P)H-nitrate reductase. NAD(P)H-nitrate reductase was shown to utilize NADPH directly as reductant. This enzyme has a preference for NADPH, but reacts about half as well with NADH.     

The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems

Malic enzyme (ME; NADP⁺-dependent; EC 1.1.40) provides NADPH for lipid biosynthesis in oleaginous microorganisms. Its role in vivo depends on there being an adequate supply of NADH to drive malate dehydrogenase to convert oxaloacetate to malate as a component of a cycle of three reactions: pyruvate → oxaloacetate → malate and, by the action of ME, back to pyruvate. However, the availability of cytosolic NADH is limited and, consequently, ancillary means of producing NADPH are necessary. Stoichiometries are given for the conversion of glucose to triacylglycerols involving ME with and without the reactions of the pentose phosphate pathway (PPP) as an additional source of NADPH. Some oleaginous microorganisms (such as Yarrowia lipolytica), however, lack a cytosolic ME and, if the PPP is the sole provider of NADPH, the theoretical yield of triacylglycerol from glucose falls to 27.6 % (w/w) from 31.6 % when ME is present. An alternative route for NADPH generation via a cytosolic isocitrate dehydrogenase (NADP⁺-dependent) is then discussed.     

Continuous monitoring of reactions that produce NADH and NADPH using immobilized luciferase and oxidoreductases from Beneckea harveyi.

Highly purified NADH and NADPH:FMN oxidoreductase and luciferase isolated from Beneckea harveyi have been immobilized to arylamine glass beads which were cemented to glass rods. The immobilized enzyme rods are stable, reuseable, and specific for either NADH or NADPH. These rods have been used to monitor reactions producing NADH or NADPH. Picomole levels of malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinase have been assayed using these rods. Glucose determination has been carried out using soluble hexokinase and glucose-6-phosphate dehydrogenase and the immobilized luciferase-oxidoreductase enzymes. Determination of ethanol concentrations as low as 0.0004% has been achieved with an immobilized alcohol dehydrogenase-NADH:FMN oxidoreductase-luciferase rod.     

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca²⁺ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.     

The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart.

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.