¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae.
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 h. 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-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-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.
Guehler Biochemistry Laboratory, Department of Chemistry, Augustana College, 639-38th Street, Rock Island, IL 61201, United States.
Yijin
Y
Tang
Alex
A
Sieg
Pamela J
PJ
Trotter
D002247:Carbon Isotopes / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D002247:Carbon Isotopes; D003470:Culture Media; D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase
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