Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies.
Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.
D000409:Alanine / Q000096:biosynthesis; D050788:Alanine Dehydrogenase; D000410:Alanine Transaminase / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism*; D001407:Bacillus / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005970:Glutamate Synthase / Q000378:metabolism; D005971:Glutamates / Q000096:biosynthesis; D018698:Glutamic Acid; D005973:Glutamine / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism; D009586:Nitrogen Fixation
D005971:Glutamates; D009566:Nitrates; D005973:Glutamine; D018698:Glutamic Acid; D009249:NADP; D000641:Ammonia; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D000410:Alanine Transaminase; D000409:Alanine