In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose-urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.

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 hours. 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, 2-13C]-acetate and analysis of carbon NMR spectra was used to examine glutamate metabolism. Incorporation of 13C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at < 15% of wild type. Analysis of 13C- enrichment of glutamate carbons indicate a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of 13C-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.

In Saccharomyces cerevisiae reductive amination of the citric acid cycle intermediate α‐ketoglutarate to glutamate is the main pathway for nitrogen assimilation. This is catalyzed by the NADP‐dependent glutamate dehydrogenases (Gdh1p & 3p), and the reverse reaction by an NAD‐dependent enzyme (Gdh2p). It has been proposed that, in GDH‐mutant strains, either the proline utilization (PUT) or the glutamine synthetase‐glutamate synthase (GS/GOGAT) pathway is the alternative nitrogen assimilation pathway to the exclusion of the other. Using a GDH‐null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway‐specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). Growth of the null mutant was only slightly impaired on all nitrogen sources tested, confirming other pathways for nitrogen assimilation exist. Enzyme assays point to GS/GOGAT as the alternate pathway when ammonia, glutamine or proline are the sole nitrogen sources. In contrast, GOGAT activity was decreased on urea and Put2p activity was slightly increased. These results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH‐dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serve as the sole compensatory pathway. (Supported by NIH grant R15‐GM069372 to P.J.T.)