Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures
NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production...
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doaj-1945013fb72d4f0a83c704548f58b7b72020-11-24T21:50:36ZengMDPI AGMetabolites2218-19892019-09-0191020510.3390/metabo9100205metabo9100205Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell CulturesEdward N. Smith0James S. O. McCullagh1R. George Ratcliffe2Nicholas J. Kruger3Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UKChemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UKDepartment of Plant Sciences, University of Oxford, Oxford OX1 3RB, UKDepartment of Plant Sciences, University of Oxford, Oxford OX1 3RB, UKNADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic <i>Arabidopsis</i> cell cultures grown on [1-<sup>2</sup>H]glucose or D<sub>2</sub>O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.https://www.mdpi.com/2218-1989/9/10/205<i>arabidopsis thaliana</i>deuteriumflavin enzymesflux analysisnadphredoxwater exchange |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Edward N. Smith James S. O. McCullagh R. George Ratcliffe Nicholas J. Kruger |
spellingShingle |
Edward N. Smith James S. O. McCullagh R. George Ratcliffe Nicholas J. Kruger Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures Metabolites <i>arabidopsis thaliana</i> deuterium flavin enzymes flux analysis nadph redox water exchange |
author_facet |
Edward N. Smith James S. O. McCullagh R. George Ratcliffe Nicholas J. Kruger |
author_sort |
Edward N. Smith |
title |
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures |
title_short |
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures |
title_full |
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures |
title_fullStr |
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures |
title_full_unstemmed |
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic <i>Arabidopsis</i> Cell Cultures |
title_sort |
limitations of deuterium-labelled substrates for quantifying nadph metabolism in heterotrophic <i>arabidopsis</i> cell cultures |
publisher |
MDPI AG |
series |
Metabolites |
issn |
2218-1989 |
publishDate |
2019-09-01 |
description |
NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic <i>Arabidopsis</i> cell cultures grown on [1-<sup>2</sup>H]glucose or D<sub>2</sub>O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes. |
topic |
<i>arabidopsis thaliana</i> deuterium flavin enzymes flux analysis nadph redox water exchange |
url |
https://www.mdpi.com/2218-1989/9/10/205 |
work_keys_str_mv |
AT edwardnsmith limitationsofdeuteriumlabelledsubstratesforquantifyingnadphmetabolisminheterotrophiciarabidopsisicellcultures AT jamessomccullagh limitationsofdeuteriumlabelledsubstratesforquantifyingnadphmetabolisminheterotrophiciarabidopsisicellcultures AT rgeorgeratcliffe limitationsofdeuteriumlabelledsubstratesforquantifyingnadphmetabolisminheterotrophiciarabidopsisicellcultures AT nicholasjkruger limitationsofdeuteriumlabelledsubstratesforquantifyingnadphmetabolisminheterotrophiciarabidopsisicellcultures |
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1725882857829695488 |