Application of 2D-TOCSY NMR to the measurement of specific 13C-enrichments in complex mixtures of 13C-labeled metabolites

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

doi:10.1016/j.ymben.2007.03.001

Cited by 48 publications

(111 citation statements)

References 18 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%

“…The temperature was 298 K. The 1D 1 H spectra were acquired by using a 30°pulse, 5,000-Hz sweep width, and 3.27-s acquisition times. 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).…”

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.

217

242

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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.

217

242

View full text Add to dashboard Cite

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“…COSY is a 1 H homonuclear shift correlation spectrum which contains information on spin coupling networks within a constituent residue through the observation of cross peaks off the diagonal [14]. The strategy of assigning a COSY spectrum is to find one unmistakably characteristic signal from which to begin the tracing of a spin system or network.…”

Section: D Cosymentioning

confidence: 99%

“…It is useful for establishing the scalar connectivity if the proton signals are from within a spin system, especially when the multiplets overlap or there is extensive second order coupling [14][15]. All protons of a coupled system are known from here.…”

Section: D Tocsymentioning

confidence: 99%