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Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction.

Zhang J, ten Pierick A, van Rossum HM, Seifar RM, Ras C, Daran JM, Heijnen JJ, Wahl SA - Sci Rep (2015)

Bottom Line: In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACT
Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate ⇄ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h(-1)) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

No MeSH data available.


The profile of Gibbs free energy (ΔrG′, kJ/mol) for the lumped G6PDH and 6PGDH reactions in the oxPPP by using cytosolic and whole cell NADPH/NADP ratio.
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f6: The profile of Gibbs free energy (ΔrG′, kJ/mol) for the lumped G6PDH and 6PGDH reactions in the oxPPP by using cytosolic and whole cell NADPH/NADP ratio.

Mentions: Here, the G6P and Ribu5P were measured (Fig. 5), and note that the Gibbs energy of reaction is not influenced by cytosolic pH because we use the sensor reaction. When S. cerevisiae was grown at pH = 5, the concentration of CO2 in equilibrium with the concentration of dissolved CO2 is in the order of 5% of the dissolved CO2 concentration, and therefore the exchange between these two pools was neglected in the calculations4243. Assuming that the concentration change of CO2(aq) (estimated of 1 mmol/L using measured off-gas CO2 levels) during the pulse is small, the ΔrG′ can be estimated by sensor reaction or whole cell NADPH/NADP ratios and metabolite measurements (Fig. 6).


Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction.

Zhang J, ten Pierick A, van Rossum HM, Seifar RM, Ras C, Daran JM, Heijnen JJ, Wahl SA - Sci Rep (2015)

The profile of Gibbs free energy (ΔrG′, kJ/mol) for the lumped G6PDH and 6PGDH reactions in the oxPPP by using cytosolic and whole cell NADPH/NADP ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525286&req=5

f6: The profile of Gibbs free energy (ΔrG′, kJ/mol) for the lumped G6PDH and 6PGDH reactions in the oxPPP by using cytosolic and whole cell NADPH/NADP ratio.
Mentions: Here, the G6P and Ribu5P were measured (Fig. 5), and note that the Gibbs energy of reaction is not influenced by cytosolic pH because we use the sensor reaction. When S. cerevisiae was grown at pH = 5, the concentration of CO2 in equilibrium with the concentration of dissolved CO2 is in the order of 5% of the dissolved CO2 concentration, and therefore the exchange between these two pools was neglected in the calculations4243. Assuming that the concentration change of CO2(aq) (estimated of 1 mmol/L using measured off-gas CO2 levels) during the pulse is small, the ΔrG′ can be estimated by sensor reaction or whole cell NADPH/NADP ratios and metabolite measurements (Fig. 6).

Bottom Line: In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACT
Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate ⇄ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h(-1)) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

No MeSH data available.