Citric acid cycle and nitrogen assimilation enzyme activities in glutamate dehydrogenase mutants of S. cerevisiae

Abstract

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.)