Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.
Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).
Duncan
P A
PA
Department of Animal Sciences, University of Illinois, Urbana-Champaign 61801.
White
B A
BA
Mackie
R I
RI
D000818:Animals; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018086:Glutamate Dehydrogenase (NADP+); D006095:Gram-Positive Cocci / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D012417:Rumen / Q000382:microbiology
D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+)