13CO2 as a universal metabolic tracer in isotopologue perturbation experiments.

A tobacco plant was illuminated for 5h in an atmosphere containing (13)CO(2) and then maintained for 10 days under standard greenhouse conditions. Nicotine, glucose, and amino acids from proteins were isolated chromatographically. Isotopologue abundances of isolated metabolites were determined quantitatively by NMR spectroscopy and mass spectrometry. The observed non-stochastic isotopologue patterns indicate (i) formation of multiply labeled photosynthetic carbohydrates during the (13)CO(2) pulse phase followed by (ii) partial catabolism of the primary photosynthetic products, and (iii) recombination of the (13)C-labeled fragments with unlabeled intermediary metabolites during the chase period. The detected and simulated isotopologue profiles of glucose and amino acids reflect carbon partitioning that is dominated by the Calvin cycle and glycolysis/glucogenesis. Retrobiosynthetic analysis of the nicotine pattern is in line with its known formation from nicotinic acid and putrescine via aspartate, glyceraldehyde phosphate and alpha-ketoglutarate as basic building blocks. The study demonstrates that pulse/chase labeling with (13)CO(2) as precursor is a powerful tool for the analysis of quantitative aspects of plant metabolism in completely unperturbed whole plants.

Robustness of central carbohydrate metabolism in developing maize kernels.

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. (13)C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-(13)C(6)]glucose or [U-(13)C(12)]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative (13)C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.

RNA DNA discrimination by the antitermination protein NusB.

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize.

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants.

Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C-13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.

Biosynthesis of benzofuran derivatives in root cultures of Tagetes patula via phenylalanine and 1-deoxy-D-xylulose 5-phosphate.

Root cultures of Tagetes patula L. cv. Carmen were grown with a mixture of unlabeled glucose and [U-(13)C(6)]glucose or [1-(13)C(1)]glucose as carbon source. Isoeuparin and (-)-4-hydroxytremetone were isolated by solvent extraction of the cultured tissue, purified by chromatography and analysed by (1)H and (13)C NMR spectroscopy. Amino acids obtained by hydrolysis of protein from the same experiments were used for the reconstruction of the labelling patterns in central metabolic intermediates. These labelling patterns were used for the prediction of isotopolog compositions in the benzofuranone derivatives via different hypothetical pathways. Comparison with the experimentally observed isotopolog distributions showed that the benzenoid ring and the acetoxy group are exclusively or predominantly (>98%) derived from phenylalanine and not from acetyl-CoA via a polyketide-type biosynthesis. The isopropylidene side chain and two carbon atoms of the furan and dihydrofuran moiety, respectively, originate from an isoprenoid building block obtained exclusively or predominantly (>98%) via the deoxyxylulose phosphate pathway. The exomethylene atom of the isopropylidene side chain is biosynthetically equivalent to the (Z)-methyl group of dimethylallyl diphosphate. The data indicate that isoeuparin and (-)-4-hydroxytremetone are assembled from 4-hydroxyacetophenone and dimethylallyl diphosphate via prenyl-substituted 4-hydroxyacetophenone and dihydrobenzofurans as intermediates.

The complex isotopologue space of glucose as a framework for the study of human intermediary metabolism.

The positional distributions of stable isotopes in metabolites provide specific fingerprints of the pathways and fluxes that have occurred in the organisms under study. In particular, modern nuclear magnetic resonance (NMR) spectroscopy enables the detailed assignment of isotope patterns in natural products, for example, in metabolites obtained from labelling experiments using (13)C-enriched precursors, such as glucose, acetate or CO2. In this study, the transient (13)C-isotopologue composition of blood glucose from an adult human volunteer after intravenous supply of [U-(13)C6]glucose was determined by high-resolution (13)C NMR spectroscopy. The non-linear progression curves displaying the relative amounts of eight (13)C-glucose isotopologues reflected the contributions of glucose metabolism by glycolytic cycling, the pentose phosphate pathway and anaplerotic reactions involving the citric acid cycle. The pilot study suggests that the experimental setting can be useful in analysing under non-invasive conditions the impact of physiological and pharmacological constraints on glucose turnover in humans.

Isotopolog perturbation techniques for metabolic networks: metabolic recycling of nutritional glucose in Drosophila melanogaster.

Drosophila melanogaster strain Oregon-R(*) was grown on standard medium supplemented with [U-(13)C(6)]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by (13)C NMR spectroscopy. All (13)C signals of the isolated glucose were multiplets arising by (13)C(13)C coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual (13)C signal patterns afforded the concentrations of certain groups of (13)C isotopologs. These data were deconvoluted by a genetic algorithm affording the abundances of all single-labeled and of 15 multiply labeled isotopologs. Among the latter group, seven isotopologs were found at concentrations >0.1 mol % with [1,2-(13)C(2)]glucose as the most prominent species. The multiply (13)C-labeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and/or passage through the pentose phosphate, the Cori cycle and/or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.