NMR-based fluxomics: Quantitative 2D NMR methods for isotopomers analysis

Massou, Stéphane; Nicolas, Cécile; Létisse, Fabien; Portais, Jean‐Charles

doi:10.1016/j.phytochem.2007.03.011

Cited by 66 publications

(89 citation statements)

References 25 publications

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“…Data were generated during steady-state 13 C-labeling experiments carried out with 13 Cenriched methanol and CO 2 at natural abundance, so that the labeling of free glycine-and glyoxylate-could be measured from the more abundant proteinogenic glycine. The 4 positional isotopomers of glycine were measured by combining 2D ZQF-TOCSY and 2D-HSQC experiments (25,26) (Table S3 and Fig. S2).…”

Section: Analysis Of Glycine Isotopomers Generated From [ 13 C]methanmentioning

confidence: 99%

“…A total of 128 scans were recorded, and relaxation delay between scans was 10 s. Proteinogenic amino acid sample was prepared as described previously (25) and modified as explained in the SI Text. Positional isotopomers of proteinogenic glycine were measured from (i) the analysis of carbon-carbon couplings in 2D-[ 1 H- 13 C]HSQC spectra and (ii) the analysis of heteronuclear 1 H, 13 C couplings in 2D ZQF-TOCSY spectra (25,26). The peak deconvolution was realized with the software GOSA-fit (www.…”

Section: Lc-ms Analysismentioning

confidence: 99%

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Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Peyraud

Kiefer

Christen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13 C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term 13 C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13 C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.13 C labeling ͉ CoA ester ͉ methylotroph ͉ one-carbon metabolism ͉ glyoxylate regeneration M ethylotrophic bacteria are organisms capable of using reduced carbon compounds, such as methanol or methane, as sole sources of carbon and energy, and they play a key role in carbon cycling in their environment. They also represent promising organisms in biotechnology for the conversion of one-carbon (C1) substrates to value-added products (1). The elucidation of the mechanisms enabling growth on reduced C1 compounds of Methylobacterium extorquens AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2-5), it is still not fully achieved. A key point has been to understand how the bacterium incorporates C1 units into cell material. The serine cycle was elucidated in this organism during the early 1960s by Quayle and coworkers (6-9). The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 co...

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Section: Analysis Of Glycine Isotopomers Generated From [ 13 C]methanmentioning

confidence: 99%

Section: Lc-ms Analysismentioning

confidence: 99%

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Peyraud

Kiefer

Christen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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217

242

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“…Cell debris was removed by ultracentrifugation, and proteins were precipitated in 70% ethanol (final concentration) and hydrolyzed in 6 M HCl for 12 h. After deuteriation, two-dimensional [ 1 H- 13 C]heteronuclear single quantum coherence spectroscopy and two-dimensional zero quantum filtered total correlation spectroscopy NMR spectra were recorded on a Bruker Avance II 500-MHz spectrometer as described (13,29,30).…”

Section: Chemicals-[mentioning

confidence: 99%

The Ethylmalonyl-CoA Pathway Is Used in Place of the Glyoxylate Cycle by Methylobacterium extorquens AM1 during Growth on Acetate

Schneider¹,

Peyraud²,

Kiefer³

et al. 2012

Journal of Biological Chemistry

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Background:The ethylmalonyl-CoA pathway represents an alternative to the glyoxylate cycle. Results: The ethylmalonyl-CoA pathway operates during acetate growth of Methylobacterium extorquens and represents one of three entry points into central metabolism. Conclusion:Isocitrate lyase-positive and -negative bacteria differ substantially in overall metabolic flux distribution. Significance: Tight coordination must exist for operation of the citric acid cycle in conjunction with the ethylmalonyl-CoA pathway.

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“…Techniques that are in use are based on NMR, [4] GC-MS [5] or LC-MS. [6] Even though MS-analysis is much more sensitive than NMR, it is not sensitive to the specific carbon atom positions. [7,8] On the other hand, the present 2D NMR methods are sensitive to the positions, [9,10] but suffer insensitivity leading to long acquisition times and, thus, to inefficiency. In principle, the local enrichments and even populations of the isotopomers are accessible directly also from the 13 C-coupled 1D 1 H NMR spectra of amino acid mixtures that can be measured in a few minutes.…”

Section: Introductionmentioning

confidence: 99%

Spectral analysis of ¹H coupled ¹³C spectra of the amino acids: adaptive spectral library of amino acid ¹³C isotopomers and positional fractional ¹³C enrichments

Tiainen

Maaheimo

Niemitz³

et al. 2007

Magnetic Reson in Chemistry

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The natural abundance 1H-coupled 13C NMR spectra of all proteogenic amino acids were measured in D2O at pH* 1. The accurate 1H,13C spin-spin coupling constants were analyzed using total-line-shape fitting. The obtained spectral parameters can be used to establish a spectral library of amino acid 13C isotopomers. The adaptive spectral library principle is introduced and discussed in this article. The simulated spectra can be applied to quantification of 13C isotopomer mixtures of amino acids and, thus, for exploring metabolic pathways. Also a protocol for amino acid 13C isotopomer metabolomic profiling in 13C labeled glucose feeding experiments is outlined. The approach is suggested to give invaluable information about positional fractional 13C enrichments, which are not easily available by any other method.

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NMR-based fluxomics: Quantitative 2D NMR methods for isotopomers analysis

Cited by 66 publications

References 25 publications

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

The Ethylmalonyl-CoA Pathway Is Used in Place of the Glyoxylate Cycle by Methylobacterium extorquens AM1 during Growth on Acetate

Spectral analysis of ¹H coupled ¹³C spectra of the amino acids: adaptive spectral library of amino acid ¹³C isotopomers and positional fractional ¹³C enrichments

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NMR-based fluxomics: Quantitative 2D NMR methods for isotopomers analysis

Cited by 66 publications

References 25 publications

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

The Ethylmalonyl-CoA Pathway Is Used in Place of the Glyoxylate Cycle by Methylobacterium extorquens AM1 during Growth on Acetate

Spectral analysis of 1H coupled 13C spectra of the amino acids: adaptive spectral library of amino acid 13C isotopomers and positional fractional 13C enrichments

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Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Spectral analysis of ¹H coupled ¹³C spectra of the amino acids: adaptive spectral library of amino acid ¹³C isotopomers and positional fractional ¹³C enrichments