NADP-Glutamate Dehydrogenase Activity Is Increased under Hyperosmotic Conditions in the Halotolerant Yeast Debaryomyces hansenii Luisa Alba-Lois, 1 Claudia Segal, 1 Beatriz Rodarte, 1 Victor Valde ´s-Lo ´pez, 1 Alexander DeLuna, 2 Rene ´ Ca ´rdenas 1 1 Departamento de Biologı ´a Celular, Facultad de Ciencias, Universidad Nacional Auto ´noma de Me ´xico, Ciudad Universitaria, 04510 Me ´xico, D.F., Me ´xico 2 Departamento de Gene ´tica Molecular, Instituto de Fisiologı ´a Celular, Universidad Nacional Auto ´noma de Me ´xico, Apartado Postal 70-242, Me ´xico, D.F. 04510, Me ´xico Received: 4 February 2003 / Accepted: 29 April 2003 Abstract. Glutamate plays an important role in osmoprotection in various bacteria. In these cases, increased intracellular glutamate pools are not attributable to the NADP-dependent glutamate dehydro- genase (NADP-GDH) or the glutamate synthase, which do not increase their activities under hyperos- motic conditions, but rather to changes in other enzymes involved in glutamate metabolism. We performed a study which indicates that, as opposed to what happens in bacteria, the activity of NADP-GDH is fivefold higher when the halotolerant yeast Debaryomyces hansenii is grown in the presence of 1 M NaCl, compared with growth in media with no added salt. Since purified NADP-GDH activity in vitro was not enhanced by the presence of salt and was more sensitive to ionic strength than the two isoenzymes from S. cerevisiae, increased enzyme synthesis is the most plausible mechanism to explain our results. We discuss the possibility that increased NADP-GDH activity in D. hansenii plays a role in counteracting the inhibitory effect of high ionic strength on the activity of this enzyme. Most free-living microorganisms possess amino acid biosynthetic pathways that allow the cell to use ammo- nium as sole nitrogen source. Ammonium utilization occurs exclusively via its incorporation into glutamate and glutamine [14]. This process can be achieved by two metabolic routes, one of them constituted by the con- certed action of glutamine synthetase and the GLT1 encoded glutamate synthase (GOGAT) [18]. The other pathway is mediated by the NADP-dependent glutamate dehydrogenase (NADP-GDH), EC 1.4.1.4, the enzyme that catalyzes the reductive amination of 2-oxoglutarate to form glutamate [12]. Most microorganisms possess a single gene encoding NADP-GDH. However, in Saccha- romyces cerevisiae, two genes (GDH1 and GDH3) have been described whose products constitute NADP-GDH isoenzymes [2, 9]. When cells are exposed to high os- molarity, they respond by accumulating and/or synthe- sizing a compatible solute inside the cell in order to counterbalance higher external osmotic pressure [7, 19]. It has been shown that in various bacteria glutamate accumulates to act as an osmolyte. When Salmonella typhimurium is grown on 500 mM NaCl, the glutamate pool increases threefold as compared with that found without salt [6, 8]. Rhizobium meliloti is also able to accumulate glutamate during osmotic stress [5]. How- ever, glutamate accumulation has not been shown to be the result of increased NADP-GDH nor GOGAT activ- ities, but it is rather due to the action of other enzymes involved in glutamate metabolism [6]. Debaryomyces hansenii is a spoilage yeast found as a contaminant of brine food; it displays the capacity to grow in media containing a wide range of salt concen- trations, including sea water, from which it has been isolated [15]. Exposition to a high environmental osmo- larity leads to dehydration of cells and decreases viabil- ity. To overcome this, cells have developed mechanisms to adapt to critical osmotic changes in their environments [3]. Osmoregulation is a complex cellular response, and many efforts have been made to understand the molec- ular mechanism of this phenomenon. In D. hansenii it Correspondence to: L. Alba-Lois; email: lal@hp.fciencias.unam.mx CURRENT MICROBIOLOGY Vol. 48 (2004), pp. 68 –72 DOI: 10.1007/s00284-003-4076-7 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2004