NADP-linked Glutamate Dehydrogenase Research Articles

TamA interacts with LeuB, the homologue of Saccharomyces cerevisiae Leu3p, to regulate gdhA expression in Aspergillus nidulans

Previous studies have shown that expression of the gdhA gene, encoding NADP-linked glutamate dehydrogenase (NADP-GDH), in Aspergillus nidulans is regulated by the major nitrogen regulatory protein AreA and its co-activator TamA. We show here that loss of TamA function has a more severe effect on the levels of gdhA expression than loss of AreA function. Using TamA as the bait in a yeast two-hybrid screen, we have identified a second protein that interacts with TamA. Sequencing analysis and functional studies have shown that this protein, designated LeuB, is a transcriptional activator with similar function to the homologous Leu3p of Saccharomyces cerevisiae. Inactivation of leuB revealed that this gene is involved in the regulation of gdhA, and an areA; leuB double mutant was shown to have similar NADP-GDH levels to a tamA single mutant. The requirement for TamA function to promote gdhA expression is likely to be due to its dual interaction with AreA and LeuB.

Evidences of high carbon catabolic enzyme activities during sporulation of Pleurotus ostreatus (Florida).

Measurements of the specific activities of the representative enzymes of different pathways linked to carbohydrate metabolism indicate that glycolysis and TCA cycles are the major route of carbohydrate catabolism in the sporulating phase of fruiting body development in Pleurotus ostreatus. Enzymes of the pentose phosphate pathway always showed lower specific activities as compared to those of the enzymes of the glycolytic pathway. The activity of NADP linked glutamate dehydrogenase which is known to be an anabolic enzyme decreased drastically in sporulating fruiting bodies and in spore containing gill tissue (spore bearing structure). Mannitol dehydrogenase activity declined significantly in the sporulating phase of P. ostreatus. The high rate of metabolism during sporulation was further supported by a lower rate of gluconeogenesis at this stage. Concentrations of all the major sugars of the fruiting body (mannitol, glucose and trehalose) decreased in the mature fruiting body and gill tissue. This indicated high catabolic activities at this stage of development.

Biochemical characterization of the yeast Yarrowia lipolytica overproducing carboxylic acids from ethanol. Nitrogen metabolism enzymes

A comparative assay of nitrogen metabolism enzymes in the Yarrowia lipolytica mutant N1 grown under conditions promoting the overproduction of either alpha-ketoglutaric acid (KGA) or citric acid showed that the overproduction of KGA correlates with an increase in the activities of the NAD- and NADP-linked glutamate dehydrogenase, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase reactions. These reactions are likely to be responsible for the overproduction of KGA by this mutant. In contrast, the overproduction of citric acid correlated with a decline in the activities of the NAD- and NADP-linked glutamate dehydrogenases and with an increase in the activities of glutamine synthetase and glutamate synthase.

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human α 2-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as α 2-macroglobulin (α2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of α2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both α-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native α2-M and PZP, respectively; (ii) limited proteolysis of bait region in α2-M and PZP, and (iii) covalent binding of cruzipain to PZP and α 2-M. Conformational and structural changes experimented by α-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain–α-M complexes were also detected by gelatin SDS–PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, α2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, α-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human α-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain. Index Descriptors and Abbreviations: Human α-macroglobulins (α-Ms); α 2-Macroglobulin (α2-M); pregnancy zone protein (PZP); Chagas' disease; T. cruzi; cysteine proteinase (CP); cruzipain; Hydrophobic membrane proteins (HMP); Isoforms of cysteine proteinases; Polyacrylamide gel electrophoresis (PAGE); NADP-linked glutamate dehydrogenase (NADP-GDH); Phenylmethanesulphonyl fluoride (PMSF); N-Benzoyl-Pro-Phe-Arg- p-nitroanilide hydrochloride (Bz-Pro-Phe-Arg-pNA); Low-density lipoprotein receptor-related protein (LRP).

Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA.

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3'-UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP-linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

Role of the regulatory gene areA of Aspergillus oryzae in nitrogen metabolism.

The areA gene of Aspergillus oryzae was cloned by cross-hybridization with the Aspergillus nidulans areA gene and was found to encode an 866-amino-acid protein that is very similar to other fungal nitrogen regulatory proteins. The A. oryzae areA gene can complement A. nidulans areA loss-of-function mutations. Functional analyses indicated that the N-terminal region of the A. oryzae AreA protein was dispensable for function and revealed a probable acidic activation domain in the protein. C-terminal truncation of the protein resulted in derepression of several nitrogen-controlled activities in A. nidulans, while deletions extending into the conserved GATA type zinc finger region abolished the activator function. The A. oryzae areA gene was inactivated by replacement with the A. oryzae pyrG gene. Strains containing the resulting areA deletion grew as well as the wild-type strain on glutamine but were unable to grow vigorously on other nitrogen sources, including ammonium. While A. oryzae exhibited reduced growth on 10 mM ammonium, the results of growth tests indicated that areA mutants of both A. oryzae and A. nidulans were affected in utilization of low concentrations of ammonium. The levels of the major nitrogen assimilatory enzymes, NADP-linked glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (EC 6.3.1.2), were determined. In both A. oryzae and A. nidulans areA mutants, the NADP-glutamate dehydrogenase levels were reduced, whereas the glutamine synthetase levels were not affected. These results suggest that the AreA protein may play an important role in the regulation of nitrogen assimilation in addition to its previously established regulatory role in nitrogen catabolism.

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression.

NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

Effect of Ferrous Ion on Amino Acid Metabolism in Mildiomycin Production by Streptoverticillium rimofaeiens.

The physiological features of the mildiomycin production by Streptoverticillium rimofaciens were examined in iron-sufficient and -deficient media. Activities of NADP-linked glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) were markedly enhanced by the addition of 10 micrograms/ml of ferrous ion into culture. Ammonium nitrogen assimilation increased with the increase in mildiomycin production. These indicate that ferrous ion contributes the supply of amino acids as a precursor of mildiomycin production. In the iron-sufficient medium, glutamate, aspartate, serine and arginine in cells were 2 to 10-fold to those in the iron-deficient medium. The major amino acid excreted from cells was arginine in the iron-sufficient culture, while in the iron-deficient culture, valine. Change in the amino acid profile by addition of ferrous ion was useful for mildiomycin biosynthesis, in which ferrous ion played a leading role in amino acid metabolism.

The anaerobic fungus Piromyces sp. strain E2: nitrogen requirement and enzymes involved in primary nitrogen metabolism.

The anaerobic fungus Piromyces sp. strain E2 appeared restricted in nitrogen utilization. Growth was only supported by ammonium as source of nitrogen. Glutamine also resulted in growth, but this was due to release of ammonia rather than to uptake and utilization of the amino acid. The fungus was not able to grow on other amino acids, albumin, urea, allantoin, or nitrate. Assimilation of ammonium is very likely to be mediated by NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS). One transaminating activity, glutamate-oxaloacetate transaminase (GOT), was demonstrated. Glutamate synthase (GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and the transaminating activity glutamate-pyruvate transaminase (GPT) were not detected in cell-free extracts of Piromyces sp. strain E2. Specific enzyme activities of both NADP-GDH and GS increased four- to sixfold under nitrogen-limiting conditions.

Partial deletion of the Saccharomyces cerevisiae GDH3 gene results in novel starvation phenotypes.

A small-scale functional analysis screen has revealed several new phenotypes associated with a large deletion of GDH3, one of two Saccharomyces cerevisiae genes known to encode NADP-linked glutamate dehydrogenase. Diploids heterozygous for the deletion are able to sporulate in rich media, while haploid deletants produce dark, wrinkled colonies containing pseudohyphal cells. The haploid cells rapidly lose viability upon starvation.

Crystallization and preliminary X-ray analysis of the NADP-specific glutamate dehydrogenase from Neurospora crassa.

The NADP-linked glutamate dehydrogenase from Neurospora crassa has been crystallized by the hanging-drop method of vapour diffusion in the presence of 0.1 M glutamate. The crystals are trigonal and are in space group P3(1)21 with unit-cell dimensions of a = b = 196.6, c = 102.0 A and with a trimer in the asymmetric unit. A full structure determination of this enzyme will lead to an understanding of the molecular basis of inter-allelic complementation observed with hybrid hexamers of naturally occurring mutants.

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants.

The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes ofTrypanosoma cruzi

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation. A and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of l-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biocbem. 192 (1990) 363–368).

Studies on saccharomyces cerevisiae cinder carbon-limiting growth transformed with plasmid pcyg4 that carries the gene for NADP-GDH

The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).

Nucleotide sequence and regulation of expression of the Aspergillus nidulans gdhA gene encoding NADP dependent glutamate dehydrogenase

The nucleotide sequence of the Aspergillus nidulans gdhA gene encoding NADP linked glutamate dehydrogenase has been determined and Northern blot analysis used to study the regulation of expression of this gene. The gdhA gene is 1485 nucleotides long and, by comparison with the corresponding Neurospora crassa am gene, has two putative introns of 53 nucleotides and a protein encoding region of 1380 nucleotides that codes for an inferred protein of 49.63 kDa which shows regions of homology with glutamate dehydrogenase proteins from a range of organisms. mRNA analysis of wild-type mycelium grown under a variety of conditions shows that: (a) the highest levels are seen with glucose as the carbon source with inorganic nitrogen; and (b) no gdhA mRNA is detectable when cells are transferred to amino acids as sole carbon source, closely matching the observed glutamate dehydrogenase activity levels under identical conditions. The results presented strongly suggest that a good carbon source is a prerequisite for transcription, but the molecular mechanism responsible is unclear.

Amino acid composition and N-terminal sequence of the NADP-linked glutamate dehydrogenase fromTrypanosoma cruzi

The NADP-linked glutamate dehydrogenase (NADP-GDH) from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified by an improved procedure. The enzyme has subunit molecular weight (47 kDa), amino acid composition and N-terminal sequence similar to those of the NADP-GDH from Escherichia coli, including the N-terminal extension of 15 amino acids present in the E. coli enzyme, but not in the NADP-GDH from Neurospora crassa.

Amino acid composition and N-terminal sequence of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

Amino acid composition and N-terminal sequence of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

Trichomonas vaginalis: Characterization of its glutamate dehydrogenase

An NADP-linked glutamate dehydrogenase (EC 1.4.1.4) was found in the soluble fraction of Trichomonas vaginalis. Its molecular weight was about 230,000 (gel filtration). The enzyme, partially purified by diafiltration and hydroxyapatite column chromatography, was heat stable (1 hr at 57 C). It catalyzed both the amination of α-ketoglutarate (mean K m 0.6 m M) and the deamination of glutamate (mean K m 1.2 m M) The optimum pH of the amination reaction was 6.7, and that of the deamination reaction was 8. Glutamate was a competitive inhibitor of the amination reaction (mean K i 5.6 m M) and α-ketoglutarate a partially competitive inhibitor of the deamination reaction (mean K i 0.45 m M). Both guanosine and inosine diphosphates (1 m M) increased the K m α-ketoglutarate fivefold (mean K i 's 0.3 and 0.4 m M, respectively). Guanosine diphosphate reduced the K m glutamate 40%. Adenosine di- and triphosphates (1 m M) were ineffective. Because the amination reaction displayed substrate inhibition, guanosine and inosine diphosphates were potent natural inhibitors, and ammonia released by deamination reactions would tend to raise pH (amination operative at acid pH), we hypothesize that the deamination reaction may predominate in the living organism.

Co-ordinate control of ammonium-scavenging enzymes in the fruit body cap ofCoprinus cinereusavoids inhibition of sporulation by ammonium

Observation of stained nuclei coupled with determination of activity of NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS) in the same tissue, showed that increase in activity of both enzymes was initiated as karyogamy became evident in normally-developing basidia of fruit bodies of the basidiomycete Coprinus cinereus. Derepression of the two enzymes was highly positively correlated (correlation coefficient 0.91), implying co-ordinated regulation. Application and injection of solutions of ammonium salts to Coprinus fruit body caps arrested further development. Other salts had no such effect, indicating a specific sensitivity to ammonium. Similar enzymic events occur during sporulation of Saccharomyces cerevisiae, which is also sensitive to inhibition by ammonium salts. It is concluded that meiosis generally includes ammonium-sensitive processes and that co-ordinated derepression of the ammonium scavenging NADP-GDH and GS is a device to ensure removal of ammonium ions from the meiotic cell.