A New Class of Glutamate Dehydrogenases (GDH)

Abstract

A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an ␣ 6 oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mM phosphate buffer, pH 7.0, at 30°C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K m , 33 mM) and sigmoidal responses to changes in ␣-ketoglutarate (S 0.5 1.3 mM; n H 1.50) and NADH (S 0.5 20 M; n H 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH 4 ؉ , by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K m for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.

The Unique Kinetic Behavior of the Very Large NAD-Dependent Glutamate Dehydrogenase fromJanthinobacterium lividum

“…A similar result was observed for Sc-GDH; the saturation profiles against -ketoglutarate and NADH showed sigmoidal responses with positive cooperativities, with Hill's constants of 1.50 and 1.52 respectively, and that against ammonium chloride was of the Michaelis-Menten type, with a K m value of 33.9 mM. 6) In these respects, the reaction manner of the reductive amination of Jl-GDH was quite similar to that of Sc-GDH.…”

“…Lower eukaryotes such as Saccharomyces serevisiae and Neurospora crassa have tetrameric GDHs with a subunit molecular mass of 115 kDa, [3][4][5] and some bacteria have GDHs with subunit molecular masses of about 180 kDa. The latter GDHs have been observed in Streptomyces clavuligerus (Sc-GDH), as a hexamer, 6) Pseudomonas aeruginosa (Pa-GDH), as a tetramer, 7) and Psychrobacter sp. TAD-1, as a dimer, 8) and represent the GDH family, which is characterized by very large subunits.…”

Streptomyces clavuligerus strain selection for clavulanic acid biosynthesis: a study based on culture composition effects and statistical analysis

“…In the presence of glycerol, AAs derived from aspartate (asparagine and threonine) and alpha-ketoglutarate (glutamine and glutamate), which are present in high concentrations in the ISP, SF and CSS media, have proved to favor CA production [16,17]. The ISP, SF and CSS media are good sources of lysine, which is known to stimulate the production of Cephamycin C rather than CA in Streptomyces clavuligerus.…”

Structural Basis for Leucine-induced Allosteric Activation of Glutamate Dehydrogenase

“…As for bacterial GDH, GDH from Clostridium symbiosum has been shown to be regulated in a homotropic manner (9). Recently, another type of GDH, GDH180, which is a very large GDH with N-and C-terminal extension domains, has been reported to be regulated heterotropically by amino acids: GDHs from Streptomyces clavuligerus (activated by aspartate and asparagine) (10), Janthinobacterium lividum (activated by aspartate and arginine) (11), and Pseudomonas aeruginosa (activated by arginine and inhibited by citrate) (12). Thus, allosteric regulation by amino acids suggests that GDH plays important roles in metabolic control at the linkage of carbon and nitrogen metabolism in various organisms.…”