Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity

Abstract

A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.Glutamate biosynthesis can be achieved through the reductive amination of 2-oxoglutarate catalyzed by NADP+-dependent glutamate dehydrogenase (NADP+-GDH) (2). In 1970, Tempest et al. (24) demonstrated the existence of an alternative pathway for glutamate biosynthesis in Klebsiella aerogenes. This pathway comprised glutamate synthase (GOGAT) and glutamine synthetase. The function of this pathway has been demonstrated in several microorganisms (3,12,23) and in higher plants (18). Physiological studies indicate that the main role of the glutamine synthetase-GOGAT pathway is in ammonium assimilation and glutamate biosynthesis under ammonium-limited conditions (23). In Neurospora crassa, GOGAT also appears to play an important role in glutamine degradation (4). N. crassa (21), Salmonella typhimurium (16), and Escherichia coli (5, 19) mutants lacking GOGAT activity have been obtained, and their characterization has contributed to the understanding of the role of GOGAT in glutamate biosynthesis.Although GOGAT activity has been previously detected in Saccharomyces cerevisiae (22), the role of GOGAT in glutamate biosynthesis and its regulation are poorly understood. Mutants altered in NADP+-GDH have been isolated from S. cerevisiae (7). The isolation of GOGAT-less mutants has been reported elsewhere (26); however, their genetic and physiological characterization has not been published, so the physiological function of GOGAT still remains obscure. Our results indicate that GOGAT plays a role in glutamate biosynthesis under conditions of ammonium excess and that under ammonium-limited conditions, both NADP+-GDH and GOGAT can participate in the synthesis of glutamate. We also present evidence suggesting that S. cerevisiae has two GOGAT activities.MATERIALS AND METHODS Strains. Table 1 describes the characteristics of the different strains used in this study.Growth conditions. Strains were routinely grown on minimal medium (MM) containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco * Corresponding author.Laboratories, Detroit, Mich.). Glucose (2% [wt/vol]) was used as the carbon source, and 40 mM (NH4)2SO4 was used as the nitrogen source. Amino acids needed to satisfy auxotrophic requirements were added at 0.01% (wt/vol). Cells wer...

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae

“…In an alternative pathway demonstrated by Tempest et al (25), glutamate is aminated to form glutamine by glutamine synthetase (GS; EC 6.3.1.2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT; EC 1.4.1.13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several microorganisms (2,13,16,23) and in higher plants (18).In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source.…”

“…In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source.…”

“…Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source. Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7).…”

“…The results indicated that plasmid insertion had occurred in the wild-type chromosomal GLT1 gene. Isolation and characterization of an ethyl methanesulfonate (EMS)-derived GDH1 mutant has been described previously (7). In order to obtain a null GDH1 mutant, the following pair of deoxyoligonucleotides was prepared: A1, 5ЈCAGAATTTCAACAAGCTT3Ј, from bp 961 to bp 978 and A2, 5ЈACCGATATCACCAGC3Ј, from bp 1410 to bp 1392 of the GDH1 gene (20,21).…”

“…Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7). Cloning of the GOGAT structural gene (GLT1) and construction of null GOGAT mutants definitively established that this yeast possesses a single NADH-GOGAT enzyme (4) and that GOGATless mutants (7) which cannot be complemented with GLT1 (unpublished results) are probably impaired in GLT1 regulation.…”