Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics.
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.
Sekiyama
Yasuyo
Y
RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi 235-0045, Japan.
Kikuchi
Jun
J
D017360:Arabidopsis / Q000254:growth & development / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010936:Plant Extracts / Q000737:chemistry; D013997:Time Factors
D002247:Carbon Isotopes; D010936:Plant Extracts; D002245:Carbon Dioxide; D005947:Glucose