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Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions.
Biotechnol Bioeng. 2008 Jul 01; 100(4):734-43.BB

Abstract

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio. Under aerobic glucose-limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(+/-14) and 320(+/-45), assuming the cytosolic pH is between 7.0 and 6.5, respectively. These values are more than 10-fold higher than the measured whole-cell total NAD/NADH ratio of 7.5(+/-2.5). Using a thermodynamic analysis of central glycolysis we demonstrate that the former are thermodynamically feasible, while the latter is not. Furthermore, we applied this novel system to study the short-term metabolic responses to perturbations. We found that the cytosolic free NAD-NADH couple became more reduced rapidly (timescale of seconds) upon a pulse of glucose (electron-donor) and that this could be reversed by the addition of acetaldehyde (electron-acceptor). In addition, these dynamics occurred without significant changes in whole-cell total NAD and NADH. This approach provides a new experimental tool for quantitative physiology and opens new possibilities in the study of energy and redox metabolism in S. cerevisiae. The same strategy should also be applicable to other microorganisms.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Canelas AB
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands.
van Gulik WM
No affiliation info available
Heijnen JJ
No affiliation info available

MeSH

AcetaldehydeBiotechnologyCytosolFructosephosphatesGlucoseGlycolysisMannitol PhosphatesNADNADPOxidation-ReductionProtein EngineeringSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsSugar Alcohol DehydrogenasesThermodynamics

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18383140

Citation

Canelas, André B., et al. "Determination of the Cytosolic Free NAD/NADH Ratio in Saccharomyces Cerevisiae Under Steady-state and Highly Dynamic Conditions." Biotechnology and Bioengineering, vol. 100, no. 4, 2008, pp. 734-43.
Canelas AB, van Gulik WM, Heijnen JJ. Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions. Biotechnol Bioeng. 2008;100(4):734-43.
Canelas, A. B., van Gulik, W. M., & Heijnen, J. J. (2008). Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions. Biotechnology and Bioengineering, 100(4), 734-43. https://doi.org/10.1002/bit.21813
Canelas AB, van Gulik WM, Heijnen JJ. Determination of the Cytosolic Free NAD/NADH Ratio in Saccharomyces Cerevisiae Under Steady-state and Highly Dynamic Conditions. Biotechnol Bioeng. 2008 Jul 1;100(4):734-43. PubMed PMID: 18383140.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions. AU - Canelas,André B, AU - van Gulik,Walter M, AU - Heijnen,Joseph J, PY - 2008/4/3/pubmed PY - 2008/8/8/medline PY - 2008/4/3/entrez SP - 734 EP - 43 JF - Biotechnology and bioengineering JO - Biotechnol Bioeng VL - 100 IS - 4 N2 - The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio. Under aerobic glucose-limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(+/-14) and 320(+/-45), assuming the cytosolic pH is between 7.0 and 6.5, respectively. These values are more than 10-fold higher than the measured whole-cell total NAD/NADH ratio of 7.5(+/-2.5). Using a thermodynamic analysis of central glycolysis we demonstrate that the former are thermodynamically feasible, while the latter is not. Furthermore, we applied this novel system to study the short-term metabolic responses to perturbations. We found that the cytosolic free NAD-NADH couple became more reduced rapidly (timescale of seconds) upon a pulse of glucose (electron-donor) and that this could be reversed by the addition of acetaldehyde (electron-acceptor). In addition, these dynamics occurred without significant changes in whole-cell total NAD and NADH. This approach provides a new experimental tool for quantitative physiology and opens new possibilities in the study of energy and redox metabolism in S. cerevisiae. The same strategy should also be applicable to other microorganisms. SN - 1097-0290 UR - https://www.unboundmedicine.com/medline/citation/18383140/Determination_of_the_cytosolic_free_NAD/NADH_ratio_in_Saccharomyces_cerevisiae_under_steady_state_and_highly_dynamic_conditions_ DB - PRIME DP - Unbound Medicine ER -