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. The GDH2-encoded NAD ؉ -dependent glutamate dehydrogenase degrades glutamate producing ammonium and ␣-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 ␣-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 ␣-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 ␣-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.

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SummaryIt is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP-glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose-grown cultures, as opposed to what has been observed for GDH1 , and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p-regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbonmediated regulation is over-imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.

In the yeast Saccharomyces cerevisiae, the paralogous genes ALT1 and ALT2 have been proposed to encode alanine aminotransferase isozymes. Although in other microorganisms this enzyme constitutes the main pathway for alanine biosynthesis, its role in S. cerevisiae had remained unclear. Results presented in this paper show that under respiratory conditions, Alt1p constitutes the sole pathway for alanine biosynthesis and catabolism, constituting the first example of an alanine aminotransferase that simultaneously carries out both functions. Conversely, under fermentative conditions, it plays a catabolic role and alanine is mainly synthesized through an alternative pathway. It can thus be concluded that ALT1 has functions in alanine biosynthesis and utilization or only alanine utilization under respiratory and fermentative conditions, respectively. ALT2 expression was repressed under all tested conditions, suggesting that Alt2p biosynthesis is strictly controlled and only allowed to express under peculiar physiological conditions.