Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe.
Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.
Perysinakis
A
A
Department of Chemistry, University of Ioannina, Greece.
Kinghorn
J R
JR
Drainas
C
C
D005970:Glutamate Synthase / Q000235:genetics / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics / Q000502:physiology*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013696:Temperature
D000644:Quaternary Ammonium Compounds; D005970:Glutamate Synthase; D005974:Glutamate-Ammonia Ligase