Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1

Abstract

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 KCI for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, at-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, SalmoneUla typhimurium, and Clostridium symbiosum revealed only 39%o amino acid homologies. The GDH from R. jfavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).

Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant

“…Curtis and coworkers purified the native GDH molecule (42) and determined by gel filtration chromatography that the molecular mass was approximately 300,000 Da (7a). This is consistent with the reported molecular masses of other hexameric GDH enzymes (12,17). With few exceptions, the subunit protein of the hexameric enzyme is in the range of 45 to 50 kDa.…”

An NAD+-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan,Entodinium caudatum

“…The cloned enzyme had optimal activity at pH 7.0 (data not shown), again similar to the bacterial enzymes (e.g. [28]). High K + concentrations, up to 0.45 M, had no influence on activity.…”

“…High K + concentrations, up to 0.45 M, had no influence on activity. Wen and Morrison [18] and Duncan et al [28] found requirements for high K + ion concentrations in ruminal bacterial GDHs.…”

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

“…Either in pure culture or in the rumen, ammonia is a preferred inorganic nitrogen source for many ruminal species including S. ruminantium D (Bryant and Robinson 1962;Karsli and Russell 2002) with glutamate dehydrogenase (GDH) and glutamine synthetase (GS) being the primary enzymes involved in ammonia assimilation (Smith et al 1981;Patterson and Hespell 1985;Duncan et al 1992;Woods and Reid 1993;Wen and Morrison 1997;Leigh and Dodsworth 2007). In contrast to other Gram-negative bacteria, S. ruminantium do not exhibit covalent modification of GS for rapid shutdown of this enzyme (Smith et al 1981;Kustu et al 1984).…”