Carbon‐13 NMR studies on the glutamate metabolism of glutamate dehydrogenase mutants of yeast

Abstract

Saccharomyces cerevisiae has three glutamate dehydrogenase enzymes encoded by the GDH1 and GDH3 (NADP+‐dependent) and GDH2 (NAD+‐dependent) genes, all of which are essential in the interconversion of alpha‐ketoglutarate, a citric acid cycle intermediate, and glutamate. In this study, strains carrying single disruptions in the GDH genes were examined. Previous studies have determined GDH activity in glucose‐grown gdh1Δ cells is <5% as compared to wild type, while activity in gdh2Δ and gdh3Δ cells is comparable to wild type. The effects of these mutations in cells pre‐grown in non‐fermentable carbon sources (acetate, raffinose, ethanol) were studied by the utilization of [1, 2‐13C] acetate, which is metabolized by the citric acid cycle and converted to glutamate. Time trials were also conducted utilizing the labeled acetate precursor. Significant enrichment of the multiplet carbon peaks in glutamate of gdh1Δ strains indicated prolonged recycling of the glutamate backbone through the citric acid cycle before conversion to glutamate. Strains carrying gdh2Δ or gdh3Δ disruptions exhibited enrichment patterns comparable to wild type cells. These data support the primacy of Gdh1p in glutamate synthesis in a non‐fermentable carbon source. (Supported by NIH‐GM069372 to P.J.T.).