Glutamate cycle, first step of nitrogen metabolism in Bacillus megaterium

Abstract

Pathways of nitrogen metabolism that lead to the formation of l-alanine, l-aspartate, l-glutamate and l-glutamine have been studied in Bacillus megaterium grown in glucose-ammonia minimal medium. The principal features of the general scheme of nitrogen metabolism, outlined in Fig. 1, are: 1 The first step, ammonia incorporation, is catalyzed by glutamine synthetase. Practically all of the nitrogen metabolized by the cell thereafter is derived from glutamine, either directly or indirectly. This conclusion is supported by three lines of evidence: (a) B. megaterium is devoid of glutamate dehydrogenase and aspartase, (b) a mutant deficient in alanine dehydrogenase grows normally in minimal medium, and (c) addition of as much as 200 μg/ml glutamine to a glucose-ammonium minimal medium is needed to enable a mutant deficient in glutamine synthetase to deplete the medium of its glucose (1 mg/ml). 2 The second step, glutamate synthesis, is catalyzed by l-glutamine (amide): 2-oxoglutarate aminotransferase (NADP oxydo-reductase), here termed glutamate synthase. In the presence of NADPH this enzyme catalyzes the formation of 2 moles of glutamate from 1 mole of glutamine and 1 mole of 2-oxoglutarate. To deplete the glucose (1 mg/ml) in a glucose-ammonia minimal medium, a mutant deficient in glutamate synthase requires the addition of 500 μg/ml glutamate. Consequently it is very likely that glutamate synthase is the only enzyme responsible for glutamate synthesis. 3 The third step, alanine and aspartate synthesis, is catalyzed by transaminases with glutamate, pyruvate and oxaloacetate as substrates. 4 The system is regulated by (a) the allosteric properties of glutamine synthetase, the activity of which is inhibited by alanine, aspartate and glutamine itself, (b) the biosynthetic control of glutamate synthase which is repressed by glutamate. The coupled reactions catalyzed by glutamine synthetase and glutamate synthase form a cycle, termed here the glutamate cycle, which plays a pivotal role in the entire cellular metabolism of nitrogen.