Functional Analysis of Ammonium Assimilation Enzymes in Neurospora crassa

Abstract

SUMMARY: In order to explore the function of each of the four enzymes involved in ammonium assimilation in Neurospora crassa [NADP-dependent glutamate dehydrogenase (GDH), NADH-dependent glutamate synthase (GOGAT) and the α and β isozymes of glutamine synthetase (GS)] growth curves, enzymic activities and intracellular pools of ammonium, glutamate and glutamine were determined in the wild-type as well as in mutants lacking one or two of these biosynthetic enzymes. These parameters were measured in ammonium-limited chemostat-type cultures, in which conditions resembling a continuous culture were achieved for the first time for a filamentous fungus, and in ammonium-excess batch cultures. NADP-dependent GDH appeared to be the main provider of cellular glutamate in the presence of limited and excess ammonium but could be partially replaced by NADH-dependent GOGAT depending upon the presence of GSβ. GOGAT appeared to have the main role in the recycling of glutamine to glutamate, particularly in ammonium-limited conditions. GSβ was the main provider of cellular glutamine but GSα could substitute for this activity if GDH was also present. In conditions of ammonium excess GDH and GOGAT were repressed by glutamate and GS was repressed by glutamine, as previously found. However, in conditions of ammonium limitation these enzymes appeared to escape nitrogen repression.