Abstract
In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.
Highlights
In the yeast Saccharomyces cerevisiae, two NADP؉-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and ␣-ketoglutarate
This study addresses the question of whether GDH1 and GDH3 play overlapping or distinct roles and whether these roles are involved in the inherent capacity of S. cerevisiae to grow under fermentative or respiratory conditions
The results presented in this paper indicate that the existence of different NADP-GDH isoforms results in glutamate biosynthesis and balanced ␣-ketoglutarate utilization
Summary
MATa GDH1 GDH3 ura leu MATa GDH1 GDH3 URA3::YIp5 LEU2::YIp351 MATa gdh1⌬::URA3 GDH3 LEU2::YIp351 MATa GDH1 gdh3⌬::LEU2 URA3::YIp5 MATa gdh1⌬::URA3 gdh3⌬::LEU2 MATa GDH1 GDH3 LEU2::YIp351 ura MATa GDH1 gdh3⌬::LEU2 ura MATa gdh1⌬::kanMX4 GDH3 LEU2::YIp351 ura MATa gdh1⌬::kanMX4 gdh3⌬::LEU2 ura. Ref. 6 Ref. 6 Ref. 6 Ref. 6 Ref. 6 This study This study This study This study that Gdh1p and Gdh3p have different allosteric properties and rates of ␣-ketoglutarate utilization. The construction of chimerical plasmids harboring combinations of the GDH1 and GDH3 promoter and coding regions allowed us to determine that expression of these two genes is differentially modulated by the carbon source. Physiological analysis of mutants lacking or overexpressing GDH1 or GDH3 showed that expression of both genes is required to achieve wild-type growth on ethanol. Our results indicate that existence of different NADP-GDH isoenzymes allows the functioning of a regulatory system in which the relative abundance of each isoform modulates the rate at which ␣-ketoglutarate is channeled to glutamate biosynthesis
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