Comparison of two glutamate producing enzymes from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The glutamate dehydrogenase from Pyrococcus</it> sp. KOD1 (Pk</it>-GDH) was purified to homogeneity and its enzymatic characteristics for glutamate production were analyzed. Pk</it>-GDH exhibited glutamate producing activity from glutamine, which was found in some higher eukaryotic GDHs. However, the k</it><inf>cat</inf>/K</it><inf>m</inf> values for ammonia and glutamine were 15×103 min−1 mM and 0.4×103 min−1 mM, respectively, indicating that the enzyme utilizes ammonia predominantly for glutamate production. Kinetic parameters of Pk</it>-GDH were compared with those of Pk</it>-GltA, which was previously identified as a glutamate synthase homologue. The turnover number for glutamine of Pk</it>-GltA (4.1×103 min−1 mM) was 10-fold higher than that of Pk</it>-GDH (0.4×103 min−1 mM). Therefore, Pk</it>-GDH is less efficient than Pk</it>-GltA for glutamate production. Pk</it>-GDH utilized both NADPH and NADH as a cofactor, Pk</it>-GltA utilized only NADPH.