NAD-glutamate Dehydrogenase Research Articles

Microalgae polysaccharides bio-stimulating effect on tomato plants: Growth and metabolic distribution

Microalgae polysaccharides represent a potentially bioressource for the enhancement and the protection of agricultural crops. We investigate the possibility to use microalgae polysaccharides as a plant biostimulant. The crude polysaccharides extract (PS) from three microalgae strains were applied to Solanum lycopersicum plants by irrigation and compared basing on their effects on shoot and root length, nodes number and shoot and root dry weight. The application of 1 mg mL−1 PS from A. platensis, D. salina and Porphorydium sp. on tomato plants improved significantly the nodes number (NN), shoot dry weight (SDW), and shoot length (SS) by75 %, 46,6 %, 25,26 % compared to control respectively. Furthermore, crude PS treatment showed an improvement of carotenoid, chlorophyll and proteins content, and Nitrate Reductase (NR), NAD-Glutamate Dehydrogenase (NAD-GDH) activities in plants leaves compared to control. 1 mg mL−1 of Porphorydium sp. enhanced significantly the carotenoid content and NAD-GDH activity by 400 %, 200 % compared to control respectively. In the same way, A. platensis PS improved chl a, chl b and NR activity by 90.1 %, 102.7 % and 88.34 compared to control respectively. In addition, it is found that a PS treatment has affected the protein content, which reaches 88.3 % under 0.5 mg mL−1 of D. salina PS treatment. GC–MS metabolomics analysis also showed a change in lipids, sterol and alkanes profiles. Some sterols precursors were increased such as Cholesta-6,22,24-triene, which may indicate an enhancement of the biosynthesis of sterols and/or steroidal glycoalkaloids in treated plants.Therefore, this is an evidence to use microalgae polysaccharides as a plant biostimulant.

Correlation-Based Network Analysis of Metabolite and Enzyme Profiles Reveals a Role of Citrate Biosynthesis in Modulating N and C Metabolism in Zea mays.

To investigate the natural variability of leaf metabolism and enzymatic activity in a maize inbred population, statistical and network analyses were employed on metabolite and enzyme profiles. The test of coefficient of variation showed that sugars and amino acids displayed opposite trends in their variance within the population, consistently with their related enzymes. The overall higher CV values for metabolites as compared to the tested enzymes are indicative for their greater phenotypic plasticity. H2 tests revealed galactinol (1) and asparagine (0.91) as the highest scorers among metabolites and nitrate reductase (0.73), NAD-glutamate dehydrogenase (0.52), and phosphoglucomutase (0.51) among enzymes. The overall low H2 scores for metabolites and enzymes are suggestive for a great environmental impact or gene-environment interaction. Correlation-based network generation followed by community detection analysis, partitioned the network into three main communities and one dyad, (i) reflecting the different levels of phenotypic plasticity of the two molecular classes as observed for the CV values and (ii) highlighting the concerted changes between classes of chemically related metabolites. Community 1 is composed mainly of enzymes and specialized metabolites, community 2′ is enriched in N-containing compounds and phosphorylated-intermediates. The third community contains mainly organic acids and sugars. Cross-community linkages are supported by aspartate, by the photorespiration amino acids glycine and serine, by the metabolically related GABA and putrescine, and by citrate. The latter displayed the strongest node-betweenness value (185.25) of all nodes highlighting its fundamental structural role in the connectivity of the network by linking between different communities and to the also strongly connected enzyme aldolase.

Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions.

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

Responses of nitrogen metabolism to copper stress in Luffa cylindrica roots

Abstract Pot experiments were performed to investigate the responses of nitrogen metabolism to copper stress in Luffa cylindrica roots. Four treatments were used, in which varying copper concentrations (25, 50, 75 and 100 μM) were added to MS medium. The fresh weights of the roots decreased gradually with the increasing copper concentrations. At the lower concentrations, the ammonium and nitrite levels increased compared with the control, but the nitrate concentrations significantly decreased with 75 and 100 μM copper. Compared with the control, nitrate reductase activity levels gradually increased with increasing copper concentrations of up to 50 μM, and nitrite reductase activity levels significantly decreased. Copper stress led to variable increases in the activities of glutamine synthetase and glutamate synthase compared with the controls. The NADH- glutamate dehydrogenase (NADH-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities were affected by the copper treatments, but that of NAD-GDH was significantly reduced by 100 μM copper. The activities of alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) showed varying changes following the copper treatments. The present results indicate that Luffa cylindrica shows altered activities of enzymes associated with nitrogen metabolism during copper stress, enabling it to monitor and adapt to changes in its N status and supply, thereby minimizing the harmful effects of the stress.

Flocculation of dimorphic yeast Benjaminiella poitrasii is altered by modulation of NAD-glutamate dehydrogenase

A strategy to control flocculation is investigated using dimorphic yeast, Benjaminiella poitrasii as a model. Parent form of this yeast (Y) exhibited faster flocculation (11.1 min) than the monomorphic yeast form mutant Y-5 (12.6 min). Atomic force microscopy revealed higher surface roughness of Y (439.34 rms) than Y-5 (52 rms). Also, the former had a zeta potential of −65.97 ± 3.45 as against −50.21 ± 2.49 for the latter. Flocculation of both Y and Y-5 could be altered by supplementing either substrates or inhibitor of NAD-glutamate dehydrogenase (NAD-GDH) in the growth media. The rate of flocculation was promoted by α-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. These interesting findings open up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing.

Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei

The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.

Tor1/2 Regulation of Retrograde Gene Expression in Saccharomyces cerevisiae Derives Indirectly as a Consequence of Alterations in Ammonia Metabolism

Retrograde genes of Saccharomyces cerevisiae encode the enzymes needed to synthesize alpha-ketoglutarate, required for ammonia assimilation, when mitochondria are damaged or non-functional because of glucose fermentation. Therefore, it is not surprising that a close association exists between control of the retrograde regulon and expression of nitrogen catabolic genes. Expression of these latter genes is nitrogen catabolite repression (NCR)-sensitive, i.e. expression is low with good nitrogen sources (e.g. glutamine) and high when only poor (e.g. proline) or limiting nitrogen sources are available. It has been reported recently that both NCR-sensitive and retrograde gene expression is negatively regulated by glutamine and induced by treating cells with the Tor1/2 inhibitor, rapamycin. These conclusions predict that NCR-sensitive and retrograde gene expression should respond in parallel to nitrogen sources, ranging from those that highly repress NCR-sensitive transcription to those that elicit minimal NCR. Because this prediction did not accommodate earlier observations that CIT2 (a retrograde gene) expression is higher in glutamine than proline containing medium, we investigated retrograde regulation further. We show that (i) retrograde gene expression correlates with intracellular ammonia and alpha-ketoglutarate generated by a nitrogen source rather than the severity of NCR it elicits, and (ii) in addition to its known regulation by NCR, NAD-glutamate dehydrogenase (GDH2) gene expression is down-regulated by ammonia under conditions where NCR is minimal. Therefore, intracellular ammonia plays a pivotal dual role, regulating the interface of nitrogen and carbon metabolism at the level of ammonia assimilation and production. Our results also indicate the effects of rapamycin treatment on CIT2 transcription, and hence Tor1/2 regulation of retrograde gene expression occur indirectly as a consequence of alterations in ammonia and glutamate metabolism.

Denaturation studies by fluorescence and quenching of thermophilic protein NAD+-glutamate dehydrogenase from Thermus thermophilus HB8.

Fluorescence techniques have been used to study the structural characteristics of many proteins. The thermophilic enzyme NAD-glutamate dehydrogenase from Thermus thermophilus HB8 is found to be a hexameric enzyme. Fluorescence spectra of native and denatured protein and effect of denaturants as urea and guanidine hydrochloride on enzyme activity of thermophilic glutamate dehydrogenase (t-GDH) have been analyzed. Native t-GDH presents the maximum emission at 338 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out.

Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications.

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

Effects of nitrogen source on growth and activity of nitrogen-assimilating enzymes in Douglas-fir seedlings

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown for 68 days in a growth chamber in nutrient solutions with ammonium, nitrate or ammonium nitrate as the nitrogen source. Among the nitrogen sources tested, whole-seedling biomass, relative growth rate (RGR), root and shoot elongation, and number of lateral roots, were greatest in seedlings grown with ammonium. In the absence of nitrogen, plant growth and formation of lateral roots were poor. Initially, glutamine synthetase, NAD-glutamate dehydrogenase and aspartate aminotransferase activities were high in young roots and shoots, but all three enzymatic activities decreased after one month of culture. In root apices, glutamine synthetase and aspartate aminotransferase activities were higher than NAD-glutamate dehydrogenase activity. Enzymatic activities were often higher in ammonium-fed seedlings than in seedings supplied with the other forms of nitrogen. Activities of all three enzymes were significantly reduced in seedlings grown in the absence of nitrogen. The beneficial effect of ammonium is discussed on the basis of its involvement in the assimilation pathways of Douglas-fir.

NAD-specific glutamate dehydrogenase fromThermus thermophilusHB8: purification and enzymatic properties

An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85–90°C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented Km values for NAD and l-glutamate of 0.27±0.03 mM and 49±10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72°C. Differential scanning calorimetry revealed a tm of 95°C for denaturation.

NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85–90°C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented Km values for NAD and l-glutamate of 0.27±0.03 mM and 49±10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72°C. Differential scanning calorimetry revealed a tm of 95°C for denaturation.

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The15N-NMR spectra of mycelia after the addition of15NH4Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.

Polymorphism in morels: isozyme electrophoretic analysis

The aim of this study was to assess whether isozyme polymorphism in different members of the Morchellaceae could be used to improve the systematics in this fungal group and to characterize intraspecific crossings between monosporal strains in Morchella esculenta. For this purpose, isozyme electrophoretic analysis of the following enzymes was performed: glutamine synthetase, NAD-glutamate dehydrogenase, NADP-glutamate dehydrogenase, aspartate aminotransferase, malate dehydrogenase, NAD-glyceraldehyde phosphate dehydrogenase, glucose phosphate isomerase, and superoxide dismutase. The analyses allowed discrimination at the inter- or intra-specific levels and could help to establish a method of identification for strains in the Morchellaceae. To a certain extent they appeared to be suitable to analyze interactions of monosporal strains of Morchella esculenta in pairing experiments. The polymorphism shown in this study was consistent with the phylogenetic relationships between the investigated strains only at the genus level.Key words: isozyme analysis, electrophoresis, Morchella sp., polymorphism.

NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids

A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified haloppilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination ofl-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+,d-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except thed-isomer of the substratel-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (KiorKa values) was correlated with the dipole moment (μ), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain.

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Hoglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as “Piceirhiza nigra” and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).

Hexokinase activity is affected in mutants of Kluyveromyces lactis resistant to glucose repression

SUMMARY: The effect of glucose on a number of mitochondrial and cytoplasmic enzymes involved in carbon metabolism (L-lactate:ferricytochrome-c 2-oxidoreductase, malate dehydrogenase, β-galactosidase, invertase, maltase and NAD-glutamate dehydrogenase) has been analysed in two different strains of Kluyveromyces lactis (PM4-4B and JA6 strains). All the above mentioned enzymes were catabolite-repressible in a strain-dependent way. From this study differences in the regulation of some enzymes have been observed between K. lactis and Saccharomyces cerevisiae. To identify genes involved in glucose metabolism that may also control carbon catabolite repression, 2-deoxyglucose-resistant mutants (Dgr+) of K. lactis that were unable to grow on glucose in the presence of antimycin A (Rag−) were selected. In this way we identified four classes of mutants. Two of them define genes previously identified: RAG1 and RAG5, encoding the low affinity glucose transport system and hexokinase, respectively. The two remaining classes of mutants define two new genes, DGR148 and DGR239, that control the level of hexokinase, indicating that in K. lactis this enzyme is positively regulated by at least two genes. All the mutants devoid of hexokinase showed relief from carbon catabolite repression of several enzymes.

The role of the NAD‐dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant

Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.

Possible involvement of cyclic adenosine 3',5'-monophosphate in the regulation of NADP-/NAD-glutamate dehydrogenase ratio and in yeast-mycelium transition of Benjaminiella poitrasii.

The effect of different adenine-containing compounds on the NADP-/NAD-glutamate dehydrogenase (GDH) ratio was studied as a function of yeast-mycelium transition in Benjaminiella poitrasii. Under in vivo conditions, at a 5.0 mM concentration, cyclic AMP (cAMP) and dibutyryl cAMP maintained the cells in the yeast form for up to 7 and 5 h, respectively, and this was reflected in the patterns of GDH ratios observed. In vitro studies of phosphorylation and dephosphorylation have also been carried out, and the results suggest a possible correlation between cAMP, the GDH ratio, and cell form in B. poitrasii.