Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics

Phytochemistry. 2007 Aug-Sep;68(16-18):2320-9. doi: 10.1016/j.phytochem.2007.04.011. Epub 2007 May 25.

Abstract

Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [(13)C(6)]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2-(13)C(1)]glucose or 10% [(13)C(6)]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the (13)C-(13)C coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated (13)C-(13)C fine couplings at the C2 position of amino acids. As another approach, f1-f3 projection of the HCACO spectrum also helps in the analysis of (13)C-(13)C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [(13)C(6)]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric (12)CO(2) assimilation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Arabidopsis / growth & development
  • Arabidopsis / metabolism*
  • Carbon Dioxide / metabolism
  • Carbon Isotopes
  • Glucose / chemistry
  • Glucose / metabolism
  • Isotope Labeling
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Plant Extracts / chemistry
  • Time Factors

Substances

  • Carbon Isotopes
  • Plant Extracts
  • Carbon Dioxide
  • Glucose