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Metabolomics-driven quantitative analysis of ammonia assimilation in E. coli.

Yuan J, Doucette CD, Fowler WU, Feng XJ, Piazza M, Rabitz HA, Wingreen NS, Rabinowitz JD - Mol. Syst. Biol. (2009)

Bottom Line: Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation.In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes.Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.

View Article: PubMed Central - PubMed

Affiliation: Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Despite extensive study of individual enzymes and their organization into pathways, the means by which enzyme networks control metabolite concentrations and fluxes in cells remains incompletely understood. Here, we examine the integrated regulation of central nitrogen metabolism in Escherichia coli through metabolomics and ordinary-differential-equation-based modeling. Metabolome changes triggered by modulating extracellular ammonium centered around two key intermediates in nitrogen assimilation, alpha-ketoglutarate and glutamine. Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes. Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.

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Elements of the nitrogen assimilation model. The ODE model describes the processes by which ammonia is taken up (N-assimilation) and nitrogen is distributed to biosynthetic pathways (N-utilization). The concentration of species within the dashed boxes (GLU, GLN, ASP, intracellular NH3, and unmodified GS and PII) are calculated as variables by the model, whereas those outside the boxes (α-KG, OA, and extracellular NH3) must be provided as inputs. N-assimilation consists of five metabolic and regulatory reactions: (1) glutamate dehydrogenase, GDH; (2) glutamine synthetase, GS; (3) glutamate synthase, GOGAT; (4) adenylyltransferase/adenylyl-removing enzyme, AT/AR; (5) uridylyltransferase/uridylyl-removing enzyme, UT/UR. Ammonia enters the cell by passive diffusion across the membrane. N-utilization consists of two different classes of reactions, which consume amino acids: those in which a nitrogen group is transferred to an acceptor molecule, recycling the carbon skeleton (N-donation) and those that consume the entire metabolite (protein and biosynthesis). In the model, the rate of GLN, GLU, and ASP consumption for biosynthesis and protein production was taken to be proportional to growth rate (for details, see Supplementary Table 5).
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f5: Elements of the nitrogen assimilation model. The ODE model describes the processes by which ammonia is taken up (N-assimilation) and nitrogen is distributed to biosynthetic pathways (N-utilization). The concentration of species within the dashed boxes (GLU, GLN, ASP, intracellular NH3, and unmodified GS and PII) are calculated as variables by the model, whereas those outside the boxes (α-KG, OA, and extracellular NH3) must be provided as inputs. N-assimilation consists of five metabolic and regulatory reactions: (1) glutamate dehydrogenase, GDH; (2) glutamine synthetase, GS; (3) glutamate synthase, GOGAT; (4) adenylyltransferase/adenylyl-removing enzyme, AT/AR; (5) uridylyltransferase/uridylyl-removing enzyme, UT/UR. Ammonia enters the cell by passive diffusion across the membrane. N-utilization consists of two different classes of reactions, which consume amino acids: those in which a nitrogen group is transferred to an acceptor molecule, recycling the carbon skeleton (N-donation) and those that consume the entire metabolite (protein and biosynthesis). In the model, the rate of GLN, GLU, and ASP consumption for biosynthesis and protein production was taken to be proportional to growth rate (for details, see Supplementary Table 5).

Mentions: To translate this qualitative understanding to quantitative equations, we wrote a differential-equation model of the network, including the cascade of covalent modification reactions that controls GS activity through (de)adenylylation (Figure 5). The model treats the extracellular concentration of ammonium and intracellular concentrations of the central carbon metabolites α-ketoglutarate and oxaloacetate as inputs to a nitrogen assimilation module, which converts these metabolites into glutamate, glutamine, and aspartate. The concentrations of glutamine and glutamate control growth rate, which in turn determines the consumption of amino acids to form biomass. The effluxes of amino acids to biomass are a known function of growth rate, dictated by the average composition of E. coli (Reitzer, 2003). These effluxes involve glutamine, glutamate, and aspartate contributing to synthesis of other metabolites (e.g., through transamination), as well as being directly assimilated into protein.


Metabolomics-driven quantitative analysis of ammonia assimilation in E. coli.

Yuan J, Doucette CD, Fowler WU, Feng XJ, Piazza M, Rabitz HA, Wingreen NS, Rabinowitz JD - Mol. Syst. Biol. (2009)

Elements of the nitrogen assimilation model. The ODE model describes the processes by which ammonia is taken up (N-assimilation) and nitrogen is distributed to biosynthetic pathways (N-utilization). The concentration of species within the dashed boxes (GLU, GLN, ASP, intracellular NH3, and unmodified GS and PII) are calculated as variables by the model, whereas those outside the boxes (α-KG, OA, and extracellular NH3) must be provided as inputs. N-assimilation consists of five metabolic and regulatory reactions: (1) glutamate dehydrogenase, GDH; (2) glutamine synthetase, GS; (3) glutamate synthase, GOGAT; (4) adenylyltransferase/adenylyl-removing enzyme, AT/AR; (5) uridylyltransferase/uridylyl-removing enzyme, UT/UR. Ammonia enters the cell by passive diffusion across the membrane. N-utilization consists of two different classes of reactions, which consume amino acids: those in which a nitrogen group is transferred to an acceptor molecule, recycling the carbon skeleton (N-donation) and those that consume the entire metabolite (protein and biosynthesis). In the model, the rate of GLN, GLU, and ASP consumption for biosynthesis and protein production was taken to be proportional to growth rate (for details, see Supplementary Table 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2736657&req=5

f5: Elements of the nitrogen assimilation model. The ODE model describes the processes by which ammonia is taken up (N-assimilation) and nitrogen is distributed to biosynthetic pathways (N-utilization). The concentration of species within the dashed boxes (GLU, GLN, ASP, intracellular NH3, and unmodified GS and PII) are calculated as variables by the model, whereas those outside the boxes (α-KG, OA, and extracellular NH3) must be provided as inputs. N-assimilation consists of five metabolic and regulatory reactions: (1) glutamate dehydrogenase, GDH; (2) glutamine synthetase, GS; (3) glutamate synthase, GOGAT; (4) adenylyltransferase/adenylyl-removing enzyme, AT/AR; (5) uridylyltransferase/uridylyl-removing enzyme, UT/UR. Ammonia enters the cell by passive diffusion across the membrane. N-utilization consists of two different classes of reactions, which consume amino acids: those in which a nitrogen group is transferred to an acceptor molecule, recycling the carbon skeleton (N-donation) and those that consume the entire metabolite (protein and biosynthesis). In the model, the rate of GLN, GLU, and ASP consumption for biosynthesis and protein production was taken to be proportional to growth rate (for details, see Supplementary Table 5).
Mentions: To translate this qualitative understanding to quantitative equations, we wrote a differential-equation model of the network, including the cascade of covalent modification reactions that controls GS activity through (de)adenylylation (Figure 5). The model treats the extracellular concentration of ammonium and intracellular concentrations of the central carbon metabolites α-ketoglutarate and oxaloacetate as inputs to a nitrogen assimilation module, which converts these metabolites into glutamate, glutamine, and aspartate. The concentrations of glutamine and glutamate control growth rate, which in turn determines the consumption of amino acids to form biomass. The effluxes of amino acids to biomass are a known function of growth rate, dictated by the average composition of E. coli (Reitzer, 2003). These effluxes involve glutamine, glutamate, and aspartate contributing to synthesis of other metabolites (e.g., through transamination), as well as being directly assimilated into protein.

Bottom Line: Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation.In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes.Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.

View Article: PubMed Central - PubMed

Affiliation: Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Despite extensive study of individual enzymes and their organization into pathways, the means by which enzyme networks control metabolite concentrations and fluxes in cells remains incompletely understood. Here, we examine the integrated regulation of central nitrogen metabolism in Escherichia coli through metabolomics and ordinary-differential-equation-based modeling. Metabolome changes triggered by modulating extracellular ammonium centered around two key intermediates in nitrogen assimilation, alpha-ketoglutarate and glutamine. Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes. Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.

Show MeSH
Related in: MedlinePlus