1, 2‐13C]‐Acetate metabolism in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Abstract

Glutamate dehydrogenases (GDH) interconvert 2‐oxoglutarate and glutamate. In yeast, NADP‐dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from 2‐oxoglutarate, while an NAD‐dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in nonfermentable acetate/raffinose (Ace/Raf) to examine the role(s) of these enzymes during aerobic metabolism. The doubling time of wt, gdh2Δ, and gdh3Δ cells was comparable at ~ 4 hours. NADP‐dependent GDH activity (Gdh1p + Gdh3p) in wt, gdh2Δ, and gdh3Δ was decreased ~80% and NAD‐dependent activity (Gdh2p) in wt and gdh3Δ was increased ~20‐fold in Ace/Raf as compared to glucose. Cells carrying the gdh1Δ allele did not grow in Ace/Raf, yet both the NADP‐dependent (Gdh3p) and NAD‐dependent (Gdh2p) GDH activity was ~3‐fold higher. Metabolism of [1, 2‐13C]‐acetate and analysis of carbon NMR spectra was used to examine glutamate metabolism. Analysis of 13C‐isotopmers and enrichment of glutamate carbons indicate a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wt. Enrichment of 13C of glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at < 15% of wt. These data suggest that Gdh1p is the primary GDH enzyme, even under nonfermentative conditions, and Gdh2p and Gdh3p play a noticeable role in glutamate metabolism. (Supported by NIH grant GM069372 [P. J. T.])