Genetic and Biochemical Characterization of Saccharomyces cerevisiae GDH1 and GDH3 encoding two NADP+-GDH isoenzymes.

DELUNA, A.; AVENDAÑO, A.; RIEGO, L.; GONZALEZ, A.

University of Maryland. College Park, MD/Washington, D.C. USA

Congreso; 1998 Yeast Genetics and Molecular Biology Meeting.; 1998

Genetics Society of America

In Saccharomyces cerevisiae glutamate is biosynthesized by the action of either NADP+-dependent glutamate dehydrogenase (GDH1) or glutamate synthase (GOGAT) (GLT1). Recently, it was found that a NADP+-GDH isozyme encoded by GDH3 constituted a third enzyme involved in glutamate biosynthesis in this yeast. Double mutants impaired in GDH1 and GLT1 were able to grow on ammonium as sole nitrogen source showing that the GDH3 encoded isozyme played a significant role in glutamate biosynthesis. The observed NADP+-GDH activity present in a gdh1, glt1 double mutant was 200-fold lower than that detected in a wild type strain, suggesting that either GDH3 is poorly expressed or that its product thas a low capacity to synthesize glutamate. Our results show that the NADP+-GDH enzyme purified from a wild type strain is composed by the GDH1 encoded isozyme. The enzyme purified from the GDH1 null mutant is more thermolabile and has considerably lower affinity for NADPH than the enzyme purified from either the wild type or the GDH3 null mutant. Antibodies raised against the enzyme purified from the wild type strain cross-reacted with extracts of either the GDH1 or GDH3 null mutants. Lac-Z fusions with GDH10rGDH3 promoters were prepared. Our results indicate that the expression of these two geries is negatively modulated by glutamate. Lac-Z fusions have been transformed into a collection of altered in Gcn4p, Gat1p, or Gln3p in order to determine whether GDH1 and GDH3 are regulation mediated by these factors.