Occurrence of Glutamine Synthetase/Glutamate Synthase Pathway in Gluconobacter suboxydans

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Abstract
The growth of Gluconobacter suboxydans was supported by addition of NH4C1 as sole nitrogen source. Glutamine synthetase and glutamate synthase activities were revealed in the cell-free extracts of NH4Cl-grown cells. The two enzymes were separated and partially purified about 120 and 200 fold, respectively. The optimum pH of glutamine synthetase was 8.0. The Km values for L-glutamine, ammonia and ATP were 6.7 mM, 3.3 mM and 0.56 mM, respectively. Mg2+ was indispensable for the activity. The enzyme activity was inhibited by glycine, L-alanine, L-hisdidine, ADP and AMP. Although glutamate synthase was strikingly unstable, it was reactivated 2~3 fold by addition of dithiothreitol. The optimum pH was 8.5. Only L-glutamine, α-ketoglutarate (α-KGA) and NADPH were the specific substrates and coenzyme. Their Km values were 0.71 mM, 2.2 µm and 20 µm, respectively. The enzyme activity was inhibited by various kinds of amino acids and α-keto acids or NADH. The effects of culture conditions on the enzyme activities were also investigated.