title: Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase.
abstract: 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 Δ(1)-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.
authors.affiliation: Guehler Biochemistry Laboratory, Department of Chemistry, Augustana College, 639-38th Street, Rock Island, IL 61201, United States.
authors.forename: Alex G
authors.initials: AG
authors.lastname: Sieg
authors.affiliation: Guehler Biochemistry Laboratory, Department of Chemistry, Augustana College, 639-38th Street, Rock Island, IL 61201, United States. Electronic address: pamtrotter@augustana.edu.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Trotter
mesh_terms: D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D005969:Glutamate Dehydrogenase / Q000172:deficiency*; D005971:Glutamates / Q000096:biosynthesis*; D005973:Glutamine / Q000378:metabolism*; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009584:Nitrogen / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005971:Glutamates; D005973:Glutamine; D011392:Proline; D005969:Glutamate Dehydrogenase; D009584:Nitrogen
keywords: Glutamate dehydrogenase; Nitrogen assimilation; Saccharomyces cerevisiae; Yeast