Retrobiosynthetic Nuclear Magnetic Resonance Analysis of Amino Acid Biosynthesis and Intermediary Metabolism. Metabolic Flux in Developing Maize Kernels

Abstract

Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.

13 C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA

“…The two flanking lines (shown in blue) arose by 13 C 13 C coupling between the observed C-2 of Glu and a directly bound 13 C atom; the coupling constant of 34.5 Hz showed that the adjacent 13 C atom causing the signal-splitting is located at position 3 (see ref. 5 for chemical shift values and 13 C 13 C coupling constants of biogenic amino acids). The relative signal intensities of the coupled signal pair indicated an abundance of 1.0 mol % for [2,3-13 C 2 ]Glu (see also Table 2, which is published as supporting information on the PNAS web site).…”

“…Samples were dissolved in 0.1 M DCl. 13 CEnrichments were determined by quantitative NMR spectroscopy (5). For this purpose, 13 C NMR spectra of the biolabeled specimens and of samples with natural 13 C abundance (i.e., with 1.1% 13 C abundance) were measured under the same experimental conditions.…”

“…In cells and organisms, such a perturbation will rapidly spread through the entire metabolic network. The quantitative analysis of that relaxation process can afford qualitative as well as quantitative information on metabolic processes in the experimental system under study (1)(2)(3)(4)(5)(6)(7).…”

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

“…In principal, these techniques can be extended toward growth of a plant organ on a general 13 C-labeled carbon source under conditions similar to those found in planta. Recently, Glawischnig et al (2001) reported initial results in this direction by labeling of maize (Zea mays) kernels.…”

Quantification of Compartmented Metabolic Fluxes in Developing Soybean Embryos by Employing Biosynthetically Directed Fractional 13C Labeling, Two-Dimensional [13C, 1H] Nuclear Magnetic Resonance, and Comprehensive Isotopomer Balancing

“…For example, Wheeler et al (1998) delineated the pathway of ascorbic acid synthesis in higher plants from 14 C labeling data, and Krook et al (1998) showed that two separate oxidative pentose phosphate pathways (oxPPP) operate in the cytosol and the plastid, using 13 C enrichment data from Daucus carota cell suspensions. More recently, a suite of articles by Eisenreich and co-workers (Bacher et al, 1999;Glawischnig et al, 2000Glawischnig et al, , 2001Glawischnig et al, , 2002 reported the abundances of isotopomers of several isolated sink metabolites in maize (Zea mays) kernels. Although these articles demonstrated advances in label measurement technology, the inferences from them were either qualitative or semiquantitative.…”