Contribution of metabolite interconversion functions to the best numerical solution of the simplified metabolic network model.

<p>Numbers of metabolite interconversion functions are indicated on x- and y-axis. The colour bar indicates the relative decrease of the cost function value, i.e. improvement of solution, when a function or a combination of functions was varied during the optimization process. All cost function values were normalised to the best of all optimization runs (100%). Functions were optimized for the simulation of the metabolic homeostasis of Col-0 under extended darkness.</p

Development of a superpathway model for primary leaf metabolism.

<p>The model structure was derived from stoichiometric and biochemical information provided by genome-scale metabolic networks and databases. The model is provided in SBML format in the supplements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092299#pone.0092299.s003" target="_blank">Model S1</a>).</p

Relative activity of the pyruvate dehydrogenase complex in Col-0 under conditions of light and extended night.

<p>Enzyme activity is given in arbitrary units which are normalised to gram fresh weight. The blue bar shows relative activity under normal light condition, the red bar shows activity under condition of extended darkness. The difference of relative activity is significant (p<0.05) and bars represent means ± SD (n  = 5).</p

Simplified model structure of the primary metabolism according to metabolic clusters.

<p>The model was derived by interconnecting the metabolic clusters by functions of interconversion (<i>f_i</i>). Clusters are named according to the description in the main text (<i>Results)</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092299#pone.0092299.s002" target="_blank">Table S1</a>.</p

Relative changes of metabolic clusters under light and extended night conditions.

<p>Bars indicate the ratios of metabolic clusters from Col-0 under extended night and light conditions. Clusters are named according to the description in the main text (<i>Results)</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092299#pone.0092299.s002" target="_blank">Table S1</a>.</p

Differential Jacobian of Col-0 under conditions of light and extended night.

<p>Bars represent the log₂-ratio of the entries in the Jacobian matrices of Col-0 under conditions of light (L) and extended night (EN), which were derived from covariance data of metabolomics data sets. Blue colour indicates a ratio >1, i.e. the Jacobian entry of the samples of extended night was higher than under normal light. White colour indicates a ratio <1, i.e. the Jacobian entry of the samples of extended night was lower than under normal light. All entries represent median values of 10³ calculations normalised to the square of interquartile distance. (<b>A</b>) and characterize the entries of the Jacobian matrix and refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092299#pone.0092299.e001" target="_blank">equation 1</a>. (<b>B</b>) shows the diagonal entries of the Jacobian matrix belonging to the metabolites described on the horizontal axis.</p

Kinetic Analysis of 14-3-3-Inhibited <i>Arabidopsis thaliana</i> Nitrate Reductase

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in kcat and a decrease in the apparent KMnitrate (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1

Kinetic Analysis of 14-3-3-Inhibited <i>Arabidopsis thaliana</i> Nitrate Reductase

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in kcat and a decrease in the apparent KMnitrate (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1

Kinetic Analysis of 14-3-3-Inhibited <i>Arabidopsis thaliana</i> Nitrate Reductase

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in kcat and a decrease in the apparent KMnitrate (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1

Kinetic Analysis of 14-3-3-Inhibited <i>Arabidopsis thaliana</i> Nitrate Reductase

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in kcat and a decrease in the apparent KMnitrate (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1