FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2046871655> ?p ?o ?g. }

• W2046871655 endingPage "43783" @default.
• W2046871655 startingPage "43775" @default.
• W2046871655 abstract "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 α-ketoglutarate. The GDH2-encoded NAD+-dependent glutamate dehydrogenase degrades glutamate producing ammonium and α-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 α-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 α-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 α-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed. 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 α-ketoglutarate. The GDH2-encoded NAD+-dependent glutamate dehydrogenase degrades glutamate producing ammonium and α-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 α-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 α-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 α-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed. NADP+-dependent glutamate dehydrogenase NAD+-dependent glutamate dehydrogenase minimal medium yeast-peptone-dextrose polymerase chain reaction N α-p-tosyl-l-lysine chloromethyl ketone 4-morpholineethanesulfonic acid base pair(s) Like most free living microorganisms, the yeast Saccharomyces cerevisiae possesses amino acid biosynthetic pathways that allow the cell to use ammonium as sole nitrogen source. Ammonium utilization occurs exclusively via its incorporation into glutamate and glutamine (1Magasanik B. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 283-317Google Scholar), a process that can be achieved by two metabolic routes. One of them is constituted by the concerted action of glutamine synthetase and the GLT1-encoded glutamate synthase (2Tempest D.W. Meers J.L. Brown C.M. Biochem. J. 1970; 117: 405-407Crossref PubMed Scopus (184) Google Scholar, 3Roon R.J. Even H.L. Larimore F. J. Bacteriol. 1974; 118: 89-95Crossref PubMed Google Scholar). The other pathway is mediated by the NADP+-dependent glutamate dehydrogenase (NADP-GDH)1 (EC 1.4.1.4), a broadly distributed enzyme that catalyzes the reductive amination of α-ketoglutarate to form glutamate (4Holzer H. Schneider S. Biochem. Z. 1957; 328: 361-367Google Scholar, 5Benachenhou-Lahfa N. Forterre P. Labendan B. J. Mol. Evol. 1993; 36: 335-346Crossref PubMed Scopus (114) Google Scholar). In S. cerevisiae, two genes (GDH1 and GDH3) have been described whose products constitute NADP-GDH isoenzymes (6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar). Glutamate catabolism is achieved through a reaction catalyzed by a different but related enzyme, the GDH2-encoded NAD+-dependent glutamate dehydrogenase (NAD-GDH) (EC 1.4.1.2), which determines glutamate degradation to ammonium and α-ketoglutarate (4Holzer H. Schneider S. Biochem. Z. 1957; 328: 361-367Google Scholar, 7Smith E.L. Boyer P.D. The Enzymes. XI. Academic Press, Inc., New York1975: 293-367Google Scholar, 8Miller S.M. Magasanik B. J. Bacteriol. 1990; 172: 4927-4935Crossref PubMed Google Scholar).S. cerevisiae is the first microorganism described in which the NADP-GDH activity is encoded by two genes (6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar); the physiological significance of this apparent redundancy is not clear. When this yeast is grown on glucose and ammonium as carbon and nitrogen sources, Gdh1p is the primary pathway for glutamate biosynthesis (6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar, 9Drillien R. Lacroute F. J. Bacteriol. 1972; 109: 203-208Crossref PubMed Google Scholar, 10Grenson M. Dubois E. Piotrowska M. Drillien R. Aigle M. Mol. Gen. Genet. 1974; 128: 73-85Crossref PubMed Scopus (66) Google Scholar). It has also been shown that GDH1 expression is regulated by the HAP system (11Dang V.D. Bohn C. Bolotin-Fukuhara M. Daignan-Fornier B. J. Bacteriol. 1996; 178: 1842-1849Crossref PubMed Google Scholar), which is known to control expression of genes involved in carbon metabolism and respiratory function (12Forsburg S.L. Guarente L. Annu. Rev. Cell Biol. 1989; 5: 153-180Crossref PubMed Scopus (129) Google Scholar). Null gdh3Δ mutants show no evident growth phenotype on glucose, and GDH3-dependent activity is negligible on this carbon source. Nevertheless, a biosynthetic role was established for GDH3 in a double gdh1Δ glt1Δ mutant that grows on ammonium sulfate as sole nitrogen source by means of Gdh3p (6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar). Moreover, global analysis of transcription suggests that GDH3 expression is influenced by the general nitrogen control system (13Cox K.H. Pinchack A.B. Cooper T.C. Yeast. 1999; 15: 703-713Crossref PubMed Scopus (55) Google Scholar).S. cerevisiae is able to grow using a variety of carbon sources under fermentative and respiratory conditions. This fact has stimulated discussion as to which specific mechanism allows α-ketoglutarate utilization for glutamate biosynthesis without impairing the integrity of the tricarboxylic acid cycle as an energy-providing system. In this regard, it has been shown that Klebsiella aerogenes strains overexpressing their gdhA gene coding for the biosynthetic NADP-GDH display an auxotrophy that is interpreted as a limitation for α-ketoglutarate and succinyl-coenzyme A (14Janes B.K. Pomposiello P.J. Perez-Matos A. Najarian D.J. Goss T.J. Bender R.A. J. Bacteriol. 2001; 183: 2709-2714Crossref PubMed Scopus (19) Google Scholar). Accordingly, α-ketoglutarate modulates NADP-GDH activity so that fluctuations in the intracellular levels of tricarboxylic acid cycle intermediates would regulate glutamate biosynthesis. Indeed, it has been shown that the signal that coordinately regulates carbon and nitrogen metabolism in Escherichia coli depends on the intracellular levels of α-ketoglutarate and glutamine (15Ninfa A. Jiang P. Atkinson M.R. Peliska J.A. Curr. Top. Cell Reg. 2000; 36: 31-75Crossref PubMed Scopus (79) Google Scholar). Interestingly, the presence of Gdh3p has been found to be increased during diauxic transition in S. cerevisiae (16Boy-Marcotte E. Perrot M. Bussereau F. Boucherie H. Jacquet M. J. Bacteriol. 1998; 180: 1044-1052Crossref PubMed Google Scholar), suggesting a particular role of this enzyme in respiratory metabolism.To understand the function of the duplicated NADP-GDH pathway present in S. cerevisiae, we purified both isoenzymes and studied their biochemical properties. Our results revealed that Gdh1p and Gdh3p have different allosteric properties and rates of α-ketoglutarate utilization. The construction of chimerical plasmids harboring combinations of the GDH1 and GDH3 promoter and coding regions allowed us to determine that expression of these two genes is differentially modulated by the carbon source. Finally, physiological analysis of mutants lacking or overexpressing GDH1 or GDH3 showed that expression of both genes is required to achieve wild-type growth on ethanol. Our results indicate that existence of different NADP-GDH isoenzymes allows the functioning of a regulatory system in which the relative abundance of each isoform modulates the rate at which α-ketoglutarate is channeled to glutamate biosynthesis.EXPERIMENTAL PROCEDURESStrainsTable I describes the characteristics of the strains used in the present work. All strains constructed for this study were LEU2 derivatives of CLA1 (ura3 leu2) and thus suited for URA3 selection. To obtain a gdh3Δ mutant, CLA1 was transformed with the BglII-linearized plasmid pLV6 (6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar) harboring a 760-bp GDH3 fragment and the yeast LEU2 gene, generating strain CLA12 (GDH1 gdh3Δ ura3). A gdh1Δ gdh3Δ ura3 mutant was obtained from CLA12, using the PCR-based gene replacement protocol described by Wach et al. (17Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2224) Google Scholar), with kanMX4 as a marker. Two deoxyoligonucleotides were designed based on the GDH1 nucleotide sequence and that of the multiple cloning site present in the pFA6a vector (17Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2224) Google Scholar). The deoxyoligonucleotide D1 (5′-CAG AAT TTC AAC AAG CTT ACG AAG AAG TTG TCT CCT CTT TGG AAGCGT ACG CTG CAG GTC GAC-3′) comprised 45 bp of the 5′ region of the GDH1 coding sequence (+11 to +55), and 18 bp (in boldface type) of the pFA6a multiple cloning site. The deoxyoligonucleotide D2 (5′-AAC ACC GAT ATC ACC AGC TGG CAC GTC AGT GTC TTG ACC AAT GTG ATC GAT GAA TTC GAG CTC G-3′) contained 45 bp corresponding to an internal GDH1 gene fragment (+451 to +495) and 19 bp (in boldface type) from the pFA6a multiple cloning site. Qiagen purified pFA6a DNA was used as template for PCR amplification in a Stratagene Robocycler 40 with the following program: one denaturing cycle for 3-min at 94 °C, followed by 26 cycles of 30-s denaturation at 94 °C, 1-min annealing at 50 °C, and 1-min extension at 72 °C. The 522-bp PCR product obtained was gel-purified and used to transform strain CLA12, generating strain CLA14. A CLA1 LEU2 derivative was obtained by transforming this strain with plasmid YIp351, generating strain CLA11. To obtain a gdh1Δ GDH3 ura3 mutant, the CLA11 strain was transformed with the above mentioned 522-bp PCR product, thus generating CLA13.Table IS. cerevisiae strains used in this workStrainRelevant genotypeSourceCLA1MAT a GDH1 GDH3 ura3 leu2Ref. 6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google ScholarCLA4MAT a GDH1 GDH3 URA3::YIp5 LEU2::YIp351Ref. 6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google ScholarCLA6MAT a gdh1Δ::URA3 GDH3 LEU2::YIp351Ref. 6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google ScholarCLA7MAT a GDH1 gdh3Δ::LEU2 URA3::YIp5Ref. 6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google ScholarCLA10MAT a gdh1Δ::URA3 gdh3Δ::LEU2Ref. 6Avendaño A. DeLuna A. Olivera H. Valenzuela L. González A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google ScholarCLA11MAT a GDH1 GDH3 LEU2::YIp351 ura3This studyCLA12MAT a GDH1 gdh3Δ::LEU2 ura3This studyCLA13MAT a gdh1Δ::kanMX4 GDH3 LEU2::YIp351 ura3This studyCLA14MAT a gdh1Δ::kanMX4 gdh3Δ::LEU2 ura3This study Open table in a new tab Yeast was transformed by the method described by Ito et al.(18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Transformants were selected for either leucine prototrophy on minimal medium (MM), or G418 resistance (200 mg/liter) (Life Technologies, Inc.) on yeast extract-peptone-dextrose (YPD)-rich medium.Growth ConditionsStrains were routinely grown on MM containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco). Filter-sterilized glucose (2%, w/v) or ethanol (2%, w/v) was used as a carbon source, and 40 mm ammonium sulfate was used as a nitrogen source. Supplements needed to satisfy auxotrophic requirements were added at 0.1 mg/ml. Cells were incubated at 30 °C with shaking (250 rpm).Construction of Low Copy Number and High Copy Number Plasmids Bearing GDH1 or GDH3 GenesAll standard molecular biology techniques were followed as previously described (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.1-7.87Google Scholar). GDH1 or GDH3 were PCR-amplified together with their 5′ promoter sequence and cloned into either the pRS316 (CEN6 ARSH4 URA3) low copy number or pRS426 (2μ ori URA3) high copy number yeast shuttle vectors (20Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar, 21Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1426) Google Scholar). For GDH1, the 2596-bp region between −952 from the start codon and +285 from the stop codon was considered to comprise the full GDH1 promoter and coding sequences (11Dang V.D. Bohn C. Bolotin-Fukuhara M. Daignan-Fornier B. J. Bacteriol. 1996; 178: 1842-1849Crossref PubMed Google Scholar). For GDH3, a 2646-bp fragment was PCR-amplified, containing the putative regulatory region (−1213 from the start codon) plus the full coding sequence and +48 from the end codon, as reported in the nucleotide sequence of chromosome I from S. cerevisiae (22Bussey H. Kaback D.B. Zhong W. Vo D.T. Clark M.W. Fortin N. Hall J. Ouellette B.F. Keng T. Barton A.B. Su Y. Davies C.J. Storms R.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3809-3813Crossref PubMed Scopus (120) Google Scholar). Deoxyoligonucleotides used for this purpose were S1 (5′-CGC GGG ATC CAG TAG TTC AGC GAC AGA AG-3′), S2 (5′-CGC GCG GAT CCC GAG TAA GGT CAT CAA TAA G-3′), S3 (5′-CGC GGG ATC CTG CGG TTA TAT GAT CTT C-3′), and S4 (5′-CGC GCG GAT CCT ACT ACA TAC ACA GAT AG-3′), generating plasmids pLAM1 (GDH1 CEN URA3), pLAM11 (GDH12μ URA3), pLAM2 (GDH3 CEN URA3), and pLAM22 (GDH3 2μURA3). DNA sequencing was carried out, using the T3/T7 priming sites of pRS316 and pRS426, at the Unidad de Biologı́a Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México (UNAM).Plasmids were subsequently transformed into CLA14 double mutant, and uracil prototrophs were selected, thus generating strains CLA14-1, CLA14-11, CLA14-2, and CLA14-22. Control strains harboring the 2μ pRS426 plasmid were constructed by transforming CLA11, CLA12, CLA13, and CLA14, generating strains CLA11-00, CLA12-00, CLA13-00, and CLA14-00, respectively.Construction of GDH1 and GDH3 Chimerical Fusion PlasmidsFusions containing either the GDH1 promoter and the GDH3 coding sequence or the GDH3 promoter and the GDH1 coding sequence were generated by overlapping PCR amplification. For this purpose, primers S1 and S5 (5′-CTC TGG TTC GCT TGT CAT TTC TTT TTC TTT TTG G-3′) were used to obtain a 980-bp product corresponding to the GDH1 5′ cognate sequence and the first 19 bp of the GDH3 coding sequence (in boldface type); this was overlapped with the 1431-bp product of primers S4 and S8 (5′-GAC AAG CGA ACC AGA GTT TC-3′), which included the complete GDH3 coding sequence. Similarly, primers S2 and S9 (5′-GAA ATT CTG GCT CTG ACA TTT TTA CTT TTT ACC-3′) were used to obtain a 1244-bp product corresponding to the GDH3 5′ cognate sequence, together with the first 17 bp of the GDH1coding sequence (in boldface type), and overlapped with the 1632-bp product of primers S3 and S10 (5′-GTC AGA GCC AGA ATT TCA AC-3′), which included the complete GDH1 coding sequence. The whole procedure led to the generation of the following plasmids: pLAM3 (5′GDH3-GDH1 CEN URA3), pLAM33 (5′GDH3-GDH1 2μ URA3), pLAM4 (5′GDH1-GDH3 CEN URA3), and pLAM44 (5′GDH1-GDH3 2μ URA3). Constructs were verified by DNA sequencing as described above.Plasmids were subsequently transformed into the CLA14 double mutant, and uracil prototrophs were selected, generating strains CLA14-3, CLA14-33, CLA14-4, and CLA14-44.NADP-GDH PurificationNADP-GDH activity was purified from ethanol-grown cultures of CLA 14-11 (gdh1Δ gdh3Δ/pLAM11 (GDH1 2μ URA3)), CLA 14-22 (gdh1Δ gdh3Δ/pLAM22 (GDH32μ URA3)), and the CLA4 wild-type strain. Strains were grown in 10 liters of MM supplemented with ethanol and ammonium sulfate, in a fermentor at the Unidad de Escalamiento, Instituto de Investigaciones Biomédicas, UNAM. Cultures were incubated at 30 °C and 300 rpm and aerated with 7 liters of oxygen/min. Cells were harvested at an optical density of 0.8–1.0 at 600 nm and stored at −70 °C until used. NADP-GDH was purified by a modified version of the method of Doherty (23Doherty D. Methods Enzymol. 1970; 17: 850-856Crossref Scopus (69) Google Scholar). All steps were carried out at 5 °C.Step 1: Whole Cell Soluble Protein ExtractCells were thawed and resuspended in 1 ml of buffer A (100 mm Tris (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 50 μg of N α-p-tosyl-l-lysine chloromethyl ketone (TLCK)/ml)/g of cells. Crude extracts were obtained after mechanical disruption of cells with a Bead-Beater (8 cycles of 1 min). After centrifugation at 30,000 × g for 30 min, protein extracts were resuspended in buffer A and diluted to ∼25 mg/ml.Step 2: Ammonium Sulfate FractionationProteins that precipitated between 40 and 65% saturation of ammonium sulfate were resuspended in buffer A. Mixtures were dialyzed twice against 4 liters of buffer B (20 mm Tris (pH 7.5), 1 mmEDTA).Step 3: DEAE Bio-Gel A ChromatographyDialyzed fractions were applied to a DEAE Bio-Gel A column (23 by 2.8 cm) equilibrated with buffer C (20 mm Tris (pH 7.5), 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 50 mg of TLCK/liter). After sample application, the column was washed with 5 column volumes of buffer B. NADP-GDH was subsequently eluted with a linear NaCl gradient of 10 column volumes (0–0.5 m). Fractions with NADP-GDH activity were pooled and dialyzed against 4 liters of buffer B.Step 4: Affinity ChromatographyA Reactive Red-agarose column (13 by 1.2 cm) was equilibrated with buffer A. After application of the sample from the previous step, the column was washed with 10 volumes of buffer A. NADP-GDH was eluted with buffer A containing 0.1 mm NADPH. Fractions with NADP-GDH activity were pooled, dialyzed against buffer B, concentrated by ultrafiltration to ∼1 mg/ml with an Amicon YM30 membrane, and stored at −70 °C until used.Enzyme Assay and Protein DeterminationWhole cell soluble protein extracts were prepared by glass bead lysis of cell pellets harvested during exponential growth, as described (24Cogoni C. Valenzuela L. González-Halphen D. Olivera H. Macino G. Ballario P. González A. J. Bacteriol. 1995; 177: 792-798Crossref PubMed Google Scholar). NADP-GDH and NAD-GDH were assayed by the method of Doherty (23Doherty D. Methods Enzymol. 1970; 17: 850-856Crossref Scopus (69) Google Scholar). One unit of activity is defined as the oxidation of 1.0 μmol of NADPH or NADH/min. Protein was measured by the method of Lowry et al. (25Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as a standard.Preparation of Anti-NADP-GDH AntibodiesAntibodies were raised in rabbits injected with purified yeast GDH1-encoded NADP-GDH and partially purified by ammonium sulfate precipitation according to the method of González-Halphen et al. (26González-Halphen D. Lindorfer M.A. Capaldi R.A. Biochemistry. 1988; 27: 7021-7031Crossref PubMed Scopus (100) Google Scholar).Electrophoresis and ImmunoblottingSDS-polyacrylamide gel electrophoresis (PAGE) and native PAGE were performed with 10 and 6% slab gels, respectively. Proteins on polyacrylamide gels were visualized with Coomassie Blue. Immunoblot analysis of SDS-electrophoresed crude extract or pure NADP-GDH was carried out as described by Towbin et al. (27Towbin H. Staehelin T. Gordon J. Bio/Technology. 1979; 24: 145-149Google Scholar). Immunoblot signaling was optimized by analyzing a number of combinations of antigen and antibody concentrations in the linear range of detectability. Scanned blots were subjected to densitometric analysis using the program ImageQuaNT 4.2 (Molecular Dynamics, Inc., Sunnyvale, CA). Data were normalized to the immunoblot signals of the corresponding purified protein.Molecular Mass DeterminationNative molecular mass was determined on a Sephacryl S-300 gel filtration column (2.6 by 90 cm) equilibrated with 50 mmTris (pH 7.5), 150 mm NaCl, and 1 mmdithiothreitol. The column was calibrated with molecular mass standards (29–700 kDa) from Sigma. Purified NADP-GDH was diluted in the same buffer, loaded into the column, and eluted at a rate of 6 ml/h. Molecular mass was determined from a plot of the log molecular mass against elution volume per void volume.The apparent molecular masses of denatured subunits were determined by SDS-PAGE with molecular mass standards (29–205 kDa) from Sigma.Amino-terminal SequencingThe isolation of polypeptides for amino-terminal sequencing was carried out as described previously (28Gutierrez-Cirlos E.B. Antaramian A. Vazquez-Acevedo M. Coria R. Gonzalez-Halphen D. J. Biol. Chem. 1994; 269: 9147-9154Abstract Full Text PDF PubMed Google Scholar). Edman degradation was carried on an Applied Biosystems Sequencer at the Laboratoire de Microséquençage des Protéines (Institut Pasteur, Paris, France).Enzyme Kinetics and Analysis of Kinetic DataNADP-GDH activity was assayed for the reductive amination reaction at different concentrations of α-ketoglutarate, NADPH, or ammonium chloride and at saturating concentrations of the remaining substrates (8 mm α-ketoglutarate, 200 μmNADPH, and 50 mm ammonium chloride). For the oxidative deamination reaction, different concentrations of glutamate or NADP+ and saturating concentration of the remaining substrate (100 mm glutamate and 300 μmNADP+) were used. The progress of the reaction was always kept below 5% conversion of the initial substrate. Measurements were made at 25 °C in 100 mm Tris at pH 7.2 or 8.0 for the reductive amination or oxidative deamination reaction, respectively. For experiments in which pH was varied, 25 mm acetic acid, 25 mm MES, 50 mm Tris was used as buffer. This buffer minimizes the change of ionic strength with pH (29Ellis K.J. Morrison J.F. Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (645) Google Scholar). Kinetic data were analyzed by nonlinear regression using the program Origin 4.1 (MicroCal Software, Inc.).Extraction and Determination of Intracellular α-KetoglutarateProtein-free cell extracts were prepared as described by Kang et al. (30Kang L. Keeler M.L. Dunlop P.C. Roon R.J. J. Bacteriol. 1982; 151: 29-35Crossref PubMed Google Scholar). The intracellular concentration of α-ketoglutarate relative to protein concentration was determined with beef glutamate dehydrogenase (Sigma) by following NADH oxidation (31Dubois E. Grenson M. Wiame J.M. Eur. J. Biochem. 1974; 48: 603-616Crossref PubMed Scopus (60) Google Scholar).Determination of Extracellular Glucose ConcentrationCells were filtered through 0.22-μm Millipore membranes. Extracellular glucose concentration was determined in the filtrate with the Glucose [HK] kit from Sigma.DISCUSSIONThis study addresses the question of whether GDH1 and GDH3 play overlapping or distinct roles and whether these roles are involved in the inherent capacity of S. cerevisiaeto grow under fermentative or respiratory conditions. The results presented in this paper indicate that the existence of different NADP-GDH isoforms results in glutamate biosynthesis and balanced α-ketoglutarate utilization. The main observations that support this assertion are the following: (a) NADP-GDHs showed differences in their allosteric properties and rates of α-ketoglutarate utilization; (b) the relative abundance of both isoenzymes depended on the nature of the carbon source; (c) a gdh3Δ mutant grew slowly on ethanol, although it had wild-type NADP-GDH activity levels (this mutant showed reduced α-ketoglutarate pools and high activity levels of the catabolic NAD-GDH, indicating an abnormal high glutamate production rate); and (d) GDH1 overexpression from a plasmid did not suppress slow growth or the reduced α-ketoglutarate pool phenotypes of a gdh1Δ gdh3Δ strain; in contrast, overexpression of GDH3 resulted in faster growth and α-ketoglutarate accumulation.It has been recently shown that the regulated expression of yeast tricarboxylic acid cycle genes is governed by two transcriptional complexes that function alternatively, depending on the integrity of the respiratory function (44Liu Z. Butow R.A. Mol. Cell. Biol. 1999; 19: 6720-6728Crossref PubMed Scopus (204) Google Scholar). The HAP system regulates the expression of genes that lead to the synthesis of α-ketoglutarate during respiratory metabolism (12Forsburg S.L. Guarente L. Annu. Rev. Cell Biol. 1989; 5: 153-180Crossref PubMed Scopus (129) Google Scholar), whereas expression of these genes is controlled by the RTG system when respiratory function is dampened or lost. This model considers that glutamate plays a central role by repressing RTG-dependent expression of genes leading to α-ketoglutarate (44Liu Z. Butow R.A. Mol. Cell. Biol. 1999; 19: 6720-6728Crossref PubMed Scopus (204) Google Scholar), thus indicating that NADP-GDH activity should be controlled accordingly. A yeast NADP-GDH activity was previously purified (32Camardella L. Di Prisco G. Garofano F. Guerrini A.M. Biochim. Biophys. Acta. 1976; 429: 324-330Crossref PubMed Scopus (11) Google Scholar) at a time when the existence of two isoenzymes was not yet recognized; thus, the kinetic properties and regulation of each isoenzyme could not possibly be discerned. In this study, purification and independent characterization of Gdh1p and Gdh3p enzymes shows that yeast possesses NADP-GDH isoforms that differ in their biochemical properties.Even after the two NADP-GDHs were recognized, induction of GDH3 could not be observed in genome-wide transcription analysis of ethanol-grown yeast, probably because of detectability limitations (45Shamji A. Kuruvilla F.G. Schreiber S.L. Curr. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 46Kuruvilla F.G. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7283-7288Crossref PubMed Scopus (69) Google Scholar). The results presented here differ from those mentioned above and show unequivocally that GDH3 expression is ethanol-induced and glucose-repressed and that GDH1expression is high on both carbon sources. This brings into accountability the role of the different NADP-GDH isoenzymes in either glucose or ethanol-grown cells. Our results also consider the allosteric regulation of the GDH3-encoded enzyme, which suggests particular regulatory properties for this activity in vivo. This would mediate a more relaxed distribution of α-ketoglutarate to either glutamate biosynthesis or energy-yielding metabolism when cells grow on a nonfermentable or limiting carbon source. During fermentative growth, glutamate biosynthesis would be afforded by the Gdh1p isoenzyme that uses α-ketoglutarate at a faster rate. Accordingly, the existence of multiple isoforms of NADP-GDH activity would provide the pacemaker mechanism that assures optimum glutamate biosynthesis in either fermentative or respiratory conditions without compromising the energy-yielding metabolism. Within this context, it is relevant that the nonfacultative yeast Kluyveromyces lactis, closely related to S. cerevisiae, bears a single homomeric NADP-GDH enzyme (47Romero M. Guzmán-León S. Aranda C. González-Halphen D. Valenzuela L. González A. Microbiology. 2000; 146: 239-245Crossref PubMed Scopus (10) Google Scholar).It has been recognized that the expression of the NAD-GDH catabolic enzyme is induced in the presence of ethanol (43Coschigano P.W. Miller S.M. Magasanik B. Mol. Cell. Biol. 1991; 11: 4455-4465Crossref PubMed Scopus (35) Google Scholar). However, the physiological significance of this observation has remained obscure, since gdh2Δ mutants show no evident phenotype in ethanol-grown cultures. Considering the results presented in this paper, it can be suggested that the coordinated action of GDH1-, GDH3-, and GDH2-encoded enzymes allows growth on ethanol, equilibrating the production and utilization of α-ke" @default.
• W2046871655 created "2016-06-24" @default.
• W2046871655 creator A5016577421 @default.
• W2046871655 creator A5035369119 @default.
• W2046871655 creator A5061235901 @default.
• W2046871655 creator A5077888801 @default.
• W2046871655 date "2001-11-01" @default.
• W2046871655 modified "2023-10-05" @default.
• W2046871655 title "NADP-Glutamate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae" @default.
• W2046871655 cites W11367174 @default.
• W2046871655 cites W1535108987 @default.
• W2046871655 cites W1547657346 @default.
• W2046871655 cites W1559382738 @default.
• W2046871655 cites W1560299240 @default.
• W2046871655 cites W1607818182 @default.
• W2046871655 cites W1637451669 @default.
• W2046871655 cites W1653847549 @default.
• W2046871655 cites W1775749144 @default.
• W2046871655 cites W1835792492 @default.
• W2046871655 cites W1890187924 @default.
• W2046871655 cites W1973782344 @default.
• W2046871655 cites W1976652073 @default.
• W2046871655 cites W1986550844 @default.
• W2046871655 cites W1988728390 @default.
• W2046871655 cites W1988737528 @default.
• W2046871655 cites W1990387319 @default.
• W2046871655 cites W1993081436 @default.
• W2046871655 cites W2015999323 @default.
• W2046871655 cites W2016534516 @default.
• W2046871655 cites W2016542380 @default.
• W2046871655 cites W2017708469 @default.
• W2046871655 cites W2027751835 @default.
• W2046871655 cites W2030213798 @default.
• W2046871655 cites W2033635262 @default.
• W2046871655 cites W2038533609 @default.
• W2046871655 cites W2051741683 @default.
• W2046871655 cites W2089844406 @default.
• W2046871655 cites W2090833124 @default.
• W2046871655 cites W2091512138 @default.
• W2046871655 cites W2100057937 @default.
• W2046871655 cites W2101699778 @default.
• W2046871655 cites W2102284511 @default.
• W2046871655 cites W2102778919 @default.
• W2046871655 cites W2106081537 @default.
• W2046871655 cites W2106645421 @default.
• W2046871655 cites W2112698071 @default.
• W2046871655 cites W2112727446 @default.
• W2046871655 cites W2139817196 @default.
• W2046871655 cites W2150904401 @default.
• W2046871655 cites W2169333620 @default.
• W2046871655 cites W2169387898 @default.
• W2046871655 cites W2172733367 @default.
• W2046871655 cites W2200991233 @default.
• W2046871655 doi "https://doi.org/10.1074/jbc.m107986200" @default.
• W2046871655 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11562373" @default.
• W2046871655 hasPublicationYear "2001" @default.
• W2046871655 type Work @default.
• W2046871655 sameAs 2046871655 @default.
• W2046871655 citedByCount "145" @default.
• W2046871655 countsByYear W20468716552012 @default.
• W2046871655 countsByYear W20468716552013 @default.
• W2046871655 countsByYear W20468716552014 @default.
• W2046871655 countsByYear W20468716552015 @default.
• W2046871655 countsByYear W20468716552016 @default.
• W2046871655 countsByYear W20468716552017 @default.
• W2046871655 countsByYear W20468716552018 @default.
• W2046871655 countsByYear W20468716552019 @default.
• W2046871655 countsByYear W20468716552020 @default.
• W2046871655 countsByYear W20468716552021 @default.
• W2046871655 countsByYear W20468716552022 @default.
• W2046871655 countsByYear W20468716552023 @default.
• W2046871655 crossrefType "journal-article" @default.
• W2046871655 hasAuthorship W2046871655A5016577421 @default.
• W2046871655 hasAuthorship W2046871655A5035369119 @default.
• W2046871655 hasAuthorship W2046871655A5061235901 @default.
• W2046871655 hasAuthorship W2046871655A5077888801 @default.
• W2046871655 hasBestOaLocation W20468716551 @default.
• W2046871655 hasConcept C119795356 @default.
• W2046871655 hasConcept C170493617 @default.
• W2046871655 hasConcept C181199279 @default.
• W2046871655 hasConcept C185592680 @default.
• W2046871655 hasConcept C2777202666 @default.
• W2046871655 hasConcept C2777576037 @default.
• W2046871655 hasConcept C2779222958 @default.
• W2046871655 hasConcept C55493867 @default.
• W2046871655 hasConcept C61174792 @default.
• W2046871655 hasConceptScore W2046871655C119795356 @default.
• W2046871655 hasConceptScore W2046871655C170493617 @default.
• W2046871655 hasConceptScore W2046871655C181199279 @default.
• W2046871655 hasConceptScore W2046871655C185592680 @default.
• W2046871655 hasConceptScore W2046871655C2777202666 @default.
• W2046871655 hasConceptScore W2046871655C2777576037 @default.
• W2046871655 hasConceptScore W2046871655C2779222958 @default.
• W2046871655 hasConceptScore W2046871655C55493867 @default.
• W2046871655 hasConceptScore W2046871655C61174792 @default.
• W2046871655 hasIssue "47" @default.
• W2046871655 hasLocation W20468716551 @default.
• W2046871655 hasOpenAccess W2046871655 @default.

• next