Glutamate Synthase from Bacillus subtilis PCI 2191

Matsuoka, Kouji; Kimura, Kinuko

doi:10.1093/oxfordjournals.jbchem.a135573

Cited by 26 publications

(19 citation statements)

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“…Assuming that there might be a similar sensitivity of the glu- tamate synthetase in E. faecalis, we defined the glutamate synthase reaction as an irreversible reaction. In addition to this, the glutamate synthase activities in E. coli and B. subtilis have both been shown to be inhibited by L-glutamate (47,48). High intracellular concentrations of L-glutamate can therefore lead to a general inhibition of the glutamate synthase activity.…”

Section: Resultsmentioning

confidence: 99%

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Veith

Solheim

Grinsven

et al. 2015

Appl Environ Microbiol

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dIncreasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially L-glutamine and L-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets. Enterococcus faecalis plays an important role in both biotechnology and medicine (1, 2). Some strains are used in the dairy industry for food fermentation and flavor production. At the same time, other strains play an increasingly important role as pathogens, especially in hospital-acquired infections (1). E. faecalis strains show multiple antibiotic resistances and are therefore the cause of serious complications. Alternative strategies for combating multiple resistant bacteria such as E. faecalis are urgently needed. Targeting vulnerable points in the bacterial metabolism could offer such an alternative route and has been discussed as a promising strategy (3, 4). However, surprisingly little is known about the detailed metabolism of E. faecalis, and it is therefore mandatory to explore this in a more elaborate fashion than previously reported.E. faecalis shows a high stress tolerance and is adapted to a variety of different native environments ranging from soil up to human or animal digestive tracts (1, 2). This environmental variability requires a highly flexible metabolic system to quickly adapt to diverse and changing environmental conditions. Therefore, strategies...

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Section: Resultsmentioning

confidence: 99%

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Veith

Solheim

Grinsven

et al. 2015

Appl Environ Microbiol

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“…nif) for N 2 fixation, and transformation of NH 3 to glutamine (Gln) by GS, a portion of which is transported to the b-proteobacterium. Cag_1339 is shown as the probable gene coding for a 2-oxoglutarate permease and Cag_0853 as probable gene coding for a glutamine transporter, both only expressed in symbiosis (Wenter et al, 2010). substrate (Mäntsälä and Zalkin, 1976;Adachi and Suzuki, 1977;Matsuoka and Kimura, 1986). In symbiosis, the increase in the intracellular concentration of 2-oxoglutarate produces a regulatory shift in nitrogen assimilation, stimulating the PII and NifA proteins and increasing GS activity.…”

Section: Is Nitrogen Assimilated Via Aladh In Free-living State?mentioning

confidence: 99%

Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in ‘Chlorochromatium aggregatum’

et al. 2013

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A symbiotic association occurs in 'Chlorochromatium aggregatum', a phototrophic consortium integrated by two species of phylogenetically distant bacteria composed by the green-sulfur Chlorobium chlorochromatii CaD3 epibiont that surrounds a central b-proteobacterium. The nonmotile chlorobia can perform nitrogen and carbon fixation, using sulfide as electron donors for anoxygenic photosynthesis. The consortium can move due to the flagella present in the central b-protobacterium. Although Chl. chlorochromatii CaD3 is never found as free-living bacteria in nature, previous transcriptomic and proteomic studies have revealed that there are differential transcription patterns between the symbiotic and free-living status of Chl. chlorocromatii CaD3 when grown in laboratory conditions. The differences occur mainly in genes encoding the enzymatic reactions involved in nitrogen and amino acid metabolism. We performed a metabolic reconstruction of Chl. chlorochromatii CaD3 and an in silico analysis of its amino acid metabolism using an elementary flux modes approach (EFM). Our study suggests that in symbiosis, Chl. chlorochromatii CaD3 is under limited nitrogen conditions where the GS/GOGAT (glutamine synthetase/glutamate synthetase) pathway is actively assimilating ammonia obtained via N 2 fixation. In contrast, when free-living, Chl. chlorochromatii CaD3 is in a condition of nitrogen excess and ammonia is assimilated by the alanine dehydrogenase (AlaDH) pathway. We postulate that 'Chlorochromatium aggregatum' originated from a parasitic interaction where the N 2 fixation capacity of the chlorobia would be enhanced by injection of 2-oxoglutarate from the b-proteobacterium via the periplasm. This consortium would have the advantage of motility, which is fundamental to a phototrophic bacterium, and the syntrophy of nitrogen and carbon sources.

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“…Kinetic analysis of the ammonia-dependent reaction of glutamate synthases from E. coli and K . aerogenes has not been reported and the interpretation of the data obtained with the enzyme from B. suhrilis (Matsuoka and Kimura, 1986) is difficult due to the nonhyperbolic kinetics observed. However, it has been suggested (Mantsala and Zalkin, 1976;Geary and Meister, 1977) that when glutamine is replaced by free ammonia, the enzyme catalyzes a glutamate-dehydrogenase-like reaction, with direct hydride transfer from NADPH to the imino acid formed upon addition of ammonia to 2-oxoglutarate ( Fig.…”

Section: ~~mentioning

confidence: 99%

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

Vanoni

Nuzzi

Rescigno

et al. 1991

European Journal of Biochemistry

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The reactions catalyzed by glutamate synthase from Azospirillum hrasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A . hrasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillurn is specific for the transfer of the 4 s hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent.The data obtained with glutamate synthase from A . hrasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillurn is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Glutamate synthase catalyzes the transfer of the amide group of L-glutamine to the C2 of 2-oxoglutarate, yielding two molecules of glutamate. The reducing equivalents are provided by reduced pyridine nucleotides in microbial glutamate synthases, or by reduced ferredoxin in photosynthetic microorganisms and in the chloroplast enzyme (Vanoni et al., 1991).Glutamate synthase is especially relevant in diazotrophs, since with glutamine synthetase, it forms the major route of ammonia assimilation at low intracellular levels of ammonia. The bacterial enzyme has been purified to homogeneity from a number of enteric bacteria, but only from two diazotrophs, namely Klehsiella aerogenes and Azospirillurn brasilense. The enzyme from A. brasilense shares several properties with that isolated from other bacterial sources (Ratti et al., 1985). It is an iron-sulfur flavoprotein, which contains 1 mol FAD, 1 mol FMN, about 8 atoms non-heme iron and about 8 atoms acidlabile sulfur/l95 kDa protomer. The protomer is formed by two dissimilar subunits of approximately 140 kDa and approximately 55 kDa for the c i and / I subunits, respectively.Bacterial glutamate synthases are specific for NADPH. Lglutamine, and 2-oxoglutarate. At alkaline pH, they can carry

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Glutamate Synthase from Bacillus subtilis PCI 2191

Cited by 26 publications

References 1 publication

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in ‘Chlorochromatium aggregatum’

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

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