NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.     

Effect of aluminium on the NAD+ kinase activity of Euglena gracilis grown heterotrophically

The effect of 2 mM AlCl3 on NAD+ kinase (E.C. 2.7.1.23) activity was studied using Euglena gracilis strain Z grown heterotrophically in darkness at pH 3.5 in the presence of lactate as sole carbon source. The Al-treatment slowed down the culture growth and suppressed the peak of NAD+ kinase activity, which characterizes the beginning of the exponential phase of growth of the control cell cultures. There are two possible explanations of the Al effect: it 1) either prevents the enzyme activation by the Ca-calmodulin (CaM) complex; or 2) suppresses the CaM-dependent NAD+ kinase form. In Euglena cells, a part of the NAD+ kinase activity is enhanced by EGTA and lowered by Ca2+: this peculiar NAD+ kinase activity is unaffected by the Al treatment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases

The disaccharide alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> O)-Octyl (1) is an acceptor for the human blood group A and B glycosyltransferases. Seven analogues of 1, containing deoxy, methoxy and arabino modifications of the Fuc residue, were chemically synthesized and kinetically evaluated in radioactive enzymatic assays. Both the enzymes tolerate modification of the 3'-OH on the fucose residue. The 2'-OH was found to be key to the recognition of the acceptors by these enzymes. The arabino derivative was recognized as an acceptor by the A transferase (Km of 200 microM), but not the B transferase and is the first synthetic acceptor capable of distinguishing between the two enzyme activities.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of coenzymically active soluble and insoluble macromolecularized NAD+ derivatives.

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an omega-carboxyalkyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

NAD kinase in equine polymorphonuclear leukocytes: properties and potential role of the enzyme

1. Subcellular fractionation of horse polymorphonuclear leukocytes revealed the exclusive location of NAD kinase in the cytosol fraction of the cells. 2. The pH optimum for the enzyme was 7.5 and the apparent Km value for NAD was 2.5 mM. 3. The kinetic parameters of NAD kinase did not change when the cells are stimulated with agents that induce a respiratory burst. 4. The enzyme was activated by Mg2+ and to a lesser extent by Ca2+. 5. NAD kinase was inhibited by EDTA, sulfhydryl reagents, NADH but not by nicotinamide. 6. The substantial phosphorylation of the intracellular NAD(H) pool noticed in stimulated granulocytes is probably due to enhanced NAD kinase activity and modulated by physiological concentrations of NADH.     

NAD+ kinase--a review

NAD+ kinase catalyzes the only (known) biochemical reaction leading to the production of NADP+ from NAD+. Most evidence indicates it is found in the cytoplasm, but reports of its presence in (other) cell bodies can not be discounted. Viewed as a protein, our knowledge of NADK composition and architecture is rudimentary. Though recognized as a large multimeric protein, no agreement is evident for the molecular weight (Mr = approximately 4-65 X 10(4] of the native protein. Is calmodulin an integral subunit of (some, all) NAD+ kinases (analogous to phosphorylase kinase in skeletal muscle)? Or is it an external modulator? Consensus is evident that a subunit of molecular weight 30-35 X 10(3) is a component of the mammalian and yeast kinase. In one case (rabbit liver) two types of subunits are reported to give rise to oligomers differing in molecular weight and catalytic activities. Viewed as an enzyme it is not known why such a complex aggregate is needed for what might otherwise appear to a routine phosphorylation reaction. Rapid equilibrium random (for pigeon liver and C. utilis preparations) and ping-pong (for A. vinelandii kinase) mechanisms have been proposed for the reaction, with multiple reactant binding sites indicated for the random cases. From the perspective of enzyme modulation, the demonstration that green plant and sea urchin egg kinases are targets for calmodulin regulation by intracellular Ca2+ links NADP+ production in these sources to the multi-level discriminatory control functions inherent to this Ca2+-protein complex. Significant questions arise from the results of various investigators considered in this review. These queries offer fertile ground for the selective design of key experiments directed to a better understanding of NAD+ kinase function and pyridine nucleotide biochemistry.     

谷氨酸棒杆菌NAD激酶的过表达对L-异亮氨酸合成的促进作用

NAD激酶催化辅酶Ⅰ[NAD(H)]发生磷酸化,转变成辅酶Ⅱ[NADP(H)],而还原态辅酶Ⅱ(NADPH)是L-异亮氨酸合成的必要辅因子。为了提高NADPH的供应,首先克隆了谷氨酸棒杆菌NAD激酶基因ppnK,并利用大肠杆菌-棒状杆菌诱导型穿梭表达载体pDXW-8和组成型穿梭表达载体pDXW-9在L-异亮氨酸合成菌——乳糖发酵短杆菌JHI3-156中进行表达。摇瓶发酵后,ppnK诱导表达菌JHI3-156/pDXW-8-ppnK的NAD激酶酶活(4.33±0.74 U/g)比pDXW-8空载菌提高了83.5%,辅酶Ⅱ与辅酶Ⅰ的比例提高了63.8%,L-异亮氨酸产量(3.86±0.12 g/L)提高了82.9%;ppnK组成表达菌JHI3-156/pDXW-9-ppnK的NAD激酶酶活(7.67±0.65 U/g)比pDXW-9空载菌提高了2.20倍,辅酶Ⅱ与辅酶Ⅰ的比例提高了1.34倍,NADPH含量提高了21.7%,L-异亮氨酸产量(2.99±0.18 g/L)提高了41.7%。这说明NAD激酶有助于辅酶Ⅱ的供应和L-异亮氨酸的生物合成,这对于其他氨基酸的生产也有一定的参考依据。     

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

NAD metabolism and mitogen stimulation of human lymphocytes

The NAD concentration in eukaryotic cells is an important parameter for many aspects of metabolism including differentiation. As reported by other workers, the NAD content of resting human peripheral blood lymphocytes was low and increased dramatically over a period of 3 days after stimulation with the mitogen phytohemagglutinin (PHA). However, simultaneous measurement of the mean cell volumes showed that the average NAD concentration in fresh quiescent lymphocytes (401 +/- 128 microM) (SD, n = 7) was similar to that observed for other cell types. Furthermore, because of the increase in cell volume which occurred on mitogen stimulation, the NAD concentration in stimulated lymphocytes was only 2-3-fold higher than in fresh resting cells. This increase was also observed in lymphocytes incubated without mitogen and was apparently due to the level of NAD precursors in the culture medium and serum supplement. Hence, the NAD concentration in resting and stimulated lymphocytes is comparable to that of other eukaryotic cells and the variations in NAD content reported earlier have been widely misinterpreted.     

Distribution and Properties of NAD Phosphorylating Reaction

Distribution of NAD phosphorylating reactions, phosphorylation through NAD kinase and phosphotransferase, was investigated. NAD kinase activity was distributed rather widely in bacteria, whereas phosphotransferase activity with p-NPP and NAD was limited to a few genera. Proteus mirabilis showed strong activity of phosphotransferase besides NAD kinase activity.

Partial purification of the phosphotransferase was attempted. The enzyme preparation possessed phosphatase activity as well as phosphotransferase activity. Phosphorylation of NAD proceeded maximally under the conditions below pH 4.0. Cu²⁺ showed stimulating effect on the activity. Besides p-NPP and phenylphosphate, various nucleotides, especially 2′ (or 3′) isomers, served as excellent phosphoryl donors, and various kinds of nucleosides and nucleotides were phosphorylated to form nucleoside monophosphates and nucleoside diphosphates.     

NAD kinases use substrate-assisted catalysis for specific recognition of NAD

Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg(2+) ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.     

Structural and functional characterization of human NAD kinase

NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.     

嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究

NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a（＋）为表达载体、E．coliBL21（DE3）为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816bp,酶分子量大约为35kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH7．5,在35qC中保温2h后仍能保持80％左右的活性。Mn2＋、Ca2＋对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4．43U／mg。动力学性质分析结果显示NAD激酶对底物NAD催化的k和圪。,分别为1．46mmol／L和0．25tzmol／（L·min）。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。     

Effects of short-term NaCl stress on calmodulin transcript levels and calmodulin-dependent NAD kinase activity in two species of tomato

NAD kinase is thought to play an important role in the plant cellular responses to biotic and abiotic stress as one of the isoforms of the enzyme is activated by the Ca^{2 +} –calmodulin (CaM) complex. NAD kinase activity was measured after short‐term NaCl stress applied to isolated cells from Lycopersicon esculentum, var. Volgogradskij (NaCl‐sensitive tomato) and L. pimpinellifolium, acc. PE2 (NaCl‐tolerant species). NAD kinase activity remained constant in the sensitive species, whereas a sharp decrease was observed in the tolerant one. After salt treatment, an induction of the calmodulin gene(s) was observed in the two species, together with a 30–50% decrease in ‘active’ CaM content, i.e. CaM able to activate purified NAD kinase, in L. pimpinellifolium. The decrease in NAD kinase activity could not, however, be fully explained by this decrease in active CaM content. A similar decrease in NAD kinase activity was also recorded with other ionic stresses and exposure to high temperatures, but not in the case of drought, exposure to low temperatures, hormonal (indole‐3‐acetic acid and abscisic acid) or H₂O₂ treatments. External Ca^{2 +} certainly plays a role in the biochemical mechanism(s) leading to NAD kinase inhibition, while no role could be shown for intracellular Ca^{2 +} . In addition, after salt stress, a modification of the redox state of NAD kinase seems to be responsible for the inhibition of the enzyme.