¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at ～4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased ～80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased ～20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was ～3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.     

BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP-dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD-dependent GDH activity was observed only in the chloroplasts. Three organelle-specific isozymes—chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2, and cytosolic/mitochondrial NADP-GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). K_m values of NADP-GDH1, NADP-GDH2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP-GDHs and one NAD-GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP-GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF-1 and BmF-2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen-assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.     

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.     

NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles

In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.     

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.     

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer’s disease, and Parkinson’s disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes γ-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.     

Glutamate catabolism in Rhizobium meliloti

The pathway by which glutamate is degraded as a carbon source has not previously been elucidated, but enzymatic analysis of Rhizobium meliloti CMF1 indicated that both glutamate dehydrogenase (GDH) and gamma-aminobutyrate (GABA) bypass activities were present in free living cells. However, when similar studies were performed on R. meliloti CMF1 bacteroids, isolated from alfalfa nodules, only GABA bypass activities were detectable. Both GDH and GABA bypass activities were influenced by the carbon source provided, with maximum activities being detected when glutamate was present as sole carbon and nitrogen source. Addition of a second carbon source, such as succinate, to the growth medium did not influence GDH activity but substantially decreased levels of the first enzyme of the GABA bypass, glutamate decarboxylase (GDC). Cyclic adenosine 3′5′-monophosphate (cAMP) failed to increase GDC activities in R. meliloti CMF1 cells grown in the presence of an additional carbon source. It is proposed that the GABA bypass is a major mechanism of glutamate carbon degradation in R. meliloti CMF1, a system whose enzymatic activities are influenced by the nature of the carbon source present in the growth environment.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

13N as a tracer for studying glutamate metabolism

This mini-review summarizes studies my associates and I carried out that are relevant to the topic of the present volume [i.e. glutamate dehydrogenase (GDH)] using radioactive ¹³N (t_1/2 9.96 min) as a biological tracer. These studies revealed the previously unrecognized rapidity with which nitrogen is exchanged among certain metabolites in vivo. For example, our work demonstrated that (a) the t_1/2 for conversion of portal vein ammonia to urea in the rat liver is ∼10-11 s, despite the need for five enzyme-catalyzed steps and two mitochondrial transport steps, (b) the residence time for ammonia in the blood of anesthetized rats is ≤7-8 s, (c) the t_1/2 for incorporation of blood-borne ammonia into glutamine in the normal rat brain is <3 s, and (d) equilibration between glutamate and aspartate nitrogen in rat liver is extremely rapid (seconds), a reflection of the fact that the components of the hepatic aspartate aminotransferase reaction are in thermodynamic equilibrium. Our work emphasizes the importance of the GDH reaction in rat liver as a conduit for dissimilating or assimilating ammonia as needed. In contrast, our work shows that the GDH reaction in rat brain appears to operate mostly in the direction of ammonia production (dissimilation). The importance of the GDH reaction as an endogenous source of ammonia in the brain and the relation of GDH to the brain glutamine cycle is discussed. Finally, our work integrates with the increasing use of positron emission tomography (PET) and nuclear magnetic resonance (NMR) to study brain ammonia uptake and brain glutamine, respectively, in normal individuals and in patients with liver disease or other diseases associated with hyperammonemia.     

Kinetics of NH(4) Assimilation in Zea mays: Preliminary Studies with a Glutamate Dehydrogenase (GDH1) Null Mutant

In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of ¹⁵NH₄⁺ assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of ¹⁵NH₄⁺ assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% (¹⁵NH₄)₂SO₄ via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of ¹⁵NH₄⁺ assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant, respectively. The lower rate of ¹⁵NH₄⁺ assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of ¹⁵N via glutamate in the GDH1-null mutant. However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate.     

Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages

Modification of lipid A is essential for bacterial adaptation to its host. Salmonella Typhimurium LpxR potentially detoxifies lipid A by 3′-O-deacylation; however, the involvement of deacylation in its adaptation remains unclear. LpxR-dependent 3′-O-deacylation was observed in the stationary phase. When macrophages were infected with stationary phase bacteria, the intracellular growth of the lpxR-null strain was lower than that of the wild-type strain. Furthermore, the expression level of inducible nitric oxide synthase was higher in the cells infected with the lpxR-null strain than in the cells infected with the wild-type strain. These results indicate that lipid A 3′-O-deacylation is beneficial for intracellular growth.     

Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate saturation albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consistent with the absence of substrate cooperativity and conformational changes associated with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides a unique opportunity to assess the contribution of allostery in metabolic regulation.     

Regulation of glutamate dehydrogenases during morphogenesis of Schizophyllum commune

The specific activities of two glutamate dehydrogenases (GDH), one requiring nicotinamide adenine dinucleotide (NAD) and the other specific for nicotinamide adenine dinucleotide phosphate (NADP), varied during growth of Schizophyllum commune as a function of the stage of the life cycle and the exogenous nitrogen source. During basidiospore germination on either glucose-NH(3) or glucose-glutamate medium, NADP-GDH increased six- to eightfold in specific activity, whereas NAD-GDH was depressed. During dikaryotic mycelial growth on either nitrogen source, the two GDH increased in a 1:1 ratio, whereas, during homokaryotic mycelial growth on glucose-NH(3), NADP-GDH activity was depressed and NAD-GDH increased six- to eightfold. Homokaryotic mycelium cultured on glucose-glutamate medium yielded high NADP-GDH activities and normal NAD-GDH activities. Intracellular NH(3) concentration and NADP-GDH activities were inversely related during spore germination and homokaryotic mycelium growth, whereas guanosine-5'-triphosphate (GTP) and l-glutamine specifically inhibited NAD- and NADP-GDH respectively in vitro. GTP inhibition was shown in extracts from cells at all stages of the life cycle. Basidiospore germling extracts contained an NADP-GDH essentially resistant to l-glutamine inhibition.     

Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

A balanced turnover of dermal fibroblasts is crucial for structural integrity and normal function of the skin. During recovery from environmental injury (such as UV exposure and physical wounding), apoptosis is an important mechanism regulating fibroblast turnover. We are interested in the role that hyaluronan (HA), an extracellular matrix molecule synthesized by HA synthase enzymes (Has), plays in regulating apoptosis in fibroblasts. We previously reported that Has1 and Has3 double knock-out (Has1/3 null) mice show accelerated wound closure and increased numbers of fibroblasts in the dermis. In the present study, we report that HA levels and Has2 mRNA expression are higher in cultured Has1/3 null primary skin fibroblasts than in wild type (WT) cells. Apoptosis induced by two different environmental stressors, UV exposure and serum starvation (SS), was reduced in the Has1/3 null cells. Hyaluronidase, added to cultures to remove extracellular HA, surprisingly had no effect upon apoptotic susceptibility to UVB or SS. However, cells treated with 4-methylumbelliferone to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. When fibroblasts were transfected with Has2-specific siRNA that lowered Has2 mRNA and HA levels by 90%, both Has1/3 null and WT cells became significantly more sensitive to apoptosis. The exogenous addition of high molecular weight HA failed to reverse this effect. We conclude that Has1/3 null skin fibroblasts (which have higher levels of Has2 gene expression) are resistant to stress-induced apoptosis.     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase