Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.     

Molecular characterization of a novel,nuclear-encoded,NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L.

Angiosperms and algae possess two distinct glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, an NAD⁺-dependent tetramer involved in cytosolic glycolysis and an NADP⁺-dependent enzyme of the Calvin cycle in chloroplasts. We have found that the gymnosperm Pinus sylvestris possesses, in addition to these, a nuclear-encoded, plastid-specific, NAD⁺-dependent GAPDH, designated GapCp, which has not previously been described from any plant. Several independent full-size cDNAs for this enzyme were isolated which encode a functional transit peptide and mature subunit very similar to that of cytosolic GAPDH of angiosperms and algae. A molecular phylogeny reveals that chloroplast GapCp and cytosolic GapC arose through gene duplication early in chlorophyte evolution. The GapCp gene is expressed as highly as that for GapC in light-grown pine seedlings. These findings suggest that aspects of compartmentalized sugar phosphate metabolism may differ in angiosperms and gymnosperms and furthermore underscore the contributions of endosymbiotic gene transfer and gene duplication to the nuclear complement of genes for enzymes of plant primary metabolism.     

The Role of Oxidized Nicotinamide Adenine Dinucleotide in Fluoride Inhibition of Active Sodium Transport in Human Erythrocytes

The rate coefficient for ²²Na release from previously labeled human erythrocytes was determined in the presence of 0.1–10 mM sodium fluoride (F). The oxidized nicotinamide adenine dinucleotide (NAD⁺) level at the end of 2 hr of incubation in tris(hydroxymethyl)aminomethane (Tris)-Ringer medium was also measured. Both parameters decreased proportionately as F concentration was raised. Both F-induced changes were immediate and were reversed by 10 mM pyruvate. The decrease in NAD⁺ concentration following enolase inhibition by F is attributed to a diminished rate of formation in the reaction catalyzed by lactic dehydrogenase (LDH) with undiminished continued utilization in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It is postulated that the NAD⁺ lowering limited the GAPDH step, resulting in proportionate decreases in the rates of phosphoglycerate kinase (PGK) and Na,K-dependent adenosine triphosphatase (Na,K-ATPase), a reaction sequence thought to link glycolysis with active Na extrusion. Adding pyruvate with F increased NAD⁺ production at the LDH step, thus reactivating GAPDH, PGK, and Na,K-ATPase and leading to the observed restoration of ²²Na release. The results suggest, therefore, that F inhibits active Na transport in intact human erythrocytes indirectly through a lowering of NAD⁺, although, direct inhibition of the Na,K-ATPase by F may possibly occur simultaneously.     

Molecular orbital study on the interaction between coenzymes and enzymes; NAD+ or NADH and dehydrogenases

The absorption band at 260 mμ of NAD⁺ shifts to 360 mg by interaction with GAPDH or its analogues. Two explanations have been given on this red shift; one is an addition of such nucleophilic residue as sulfhydryl group in the enzyme to the position four in nicotinamide nucleus of NAD⁺, and the other is the charge transfer from such aromatic amino acid as tryptophan to NAD⁺. In the present paper, possibility of the charge transfer from indole residue to NAD⁺ was investigated quantum chemically. Taking into account of the electric field due to the charges in the enzyme, the absorption band of the NAD⁺-enzyme complex at 360 mμ was explained as a charge transfer from indole nucleus to NAD⁺. The blue shift of the absorption band of NADH at 340 mμ was also explained by taking into account of the electric field and this supported the proposition of Kosower (1962a).Stacking of adenine nucleus with indole nucleus in the NAD⁺-enzyme complex was suggested from the NMR spectroscopic data. Our molecular orbital calculations predicted that the effects of adenine on spectral shifts were not significant.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD⁺. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD⁺, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD⁺ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD⁺ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD⁺ precursors, or a combination of KP inhibitors with NAD⁺ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD⁺ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD⁺ metabolism can synergistically increase life span.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Studies on the effect of NAD(H) on nitrogenase activity in Rhodospirillum rubrum

The effect of NAD(P) and analogs of this nucleotide on nitrogenase activity in Rhodospirillum rubrum has been studied. Addition of NAD⁺ to nitrogen fixing Rsp. rubrum leads to inhibition of nitrogenase. NADP⁺ has the same effect but NADH or analogs modified in the nicotinamide portion do not cause inhibition. In contrast to ammonium ions, addition of NAD⁺ leads to inhibition of nitrogenase in cells that have been N-starved under argon. The inhibitory effect of NAD⁺ is more pronounced at lower light intensities. Addition of NAD⁺ also leads to inhibition of glutamine synthetase, a phenomenon also occurring when “switchoff” is produced by the addition of effectors such as ammonium ions or glutamine. It is also shown that NAD⁺ is taken up by Rsp. rubrum cells.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD⁺ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi‐bi ordered mechanism in which NAD⁺ binds to the enzyme before the aldehyde. Previous studies showed that NAD⁺ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD⁺ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD⁺ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD⁺. The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD⁺ to the enzyme caused changes in its secondary structure, whereas the presence of K⁺ helps maintain its α‐helix content. K⁺ increased the thermal stability of the pkBADH‐NAD⁺ complex by 5.3°C. ITC data showed that NAD⁺ binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD⁺ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.     

Metabolism of acetaldehyde and Custers effect in the yeast

Brettanomyces abstinens growing on different initial glucose concentrations showed an anaerobic inhibition of fermentation. This Custers effect decreased as the initial glucose concentration in the medium increased. Two aldehyde dehydrogenases, one NAD⁺-linked and the other NADP⁺-linked were observed. The results suggest that the NAD⁺-linked enzyme is involved in the production of acetic acid and is repressed by glucose. The NADP⁺-linked enzyme seems to be a constitutive enzyme. Acetyl-CoA synthetase activity also was not greatly affected by the growth conditions.The results support the earlier hypothesis that the Custers effect in Brettanomyces is provoked by the reduction of NAD⁺ in the conversion of acetaldehyde to acetic acid.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

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.