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. 2009 Mar 24;106(12):4846-51.
doi: 10.1073/pnas.0810932106. Epub 2009 Mar 4.

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

Rémi Peyraud  1 , Patrick Kiefer, Philipp Christen, Stephane Massou, Jean-Charles Portais, Julia A Vorholt
Affiliations

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

Rémi Peyraud et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Pathways proposed for glyoxylate regeneration in isocitrate lyase-negative bacteria. The reactions that are specific to the GRC (20) or to the EMCP (23) are indicated. For designations of genes and enzymes, see Table S1. Metabolite numbers are according to Fig. 2.
Fig. 2.
Fig. 2.
LC-MS analysis of CoA thioesters occurring in cell extracts of M. extorquens AM1 during growth on methanol. 1, malyl-CoA; 2, succinyl-CoA; 3, methylmalonyl-CoA; 4, β-methylmalyl-CoA; 5, CoA; 6, mesaconyl-CoA; 7, methylsuccinyl-CoA; 8, ethylmalonyl-CoA; 9, acetyl-CoA; 10, 3-hydroxybutyryl-CoA; 11, propionyl-CoA; 12, crotonyl-CoA; and 13, butyryl-CoA.
Fig. 3.
Fig. 3.
Predicted fate of carbons from [1-13C]acetate to propionyl-CoA. (A) GRC. (B) EMCP. Black triangles indicate carbon derived from the first carbon of acetate; gray circles, carbon derived from CO2.
Fig. 4.
Fig. 4.
Kinetics of 13C label incorporation in CoA thioesters after addition of [1-13C]acetate to [12C]methanol-grown M. extorquens AM1 cells. L1-Ac represents the percent of 13C label incorporated in a given metabolite, normalized to the maximal number of carbon atoms received from the first carbon of acetate. Results are mean values + SDs from 3 independent biological replicates.
Fig. 5.
Fig. 5.
Comparison of the time course evolution of M+2 isotopomeric fractions in propionyl-CoA (○) and methylsuccinyl-CoA (●) during [1-13C]acetate labeling experiments carried out with methanol-grown M. extorquens AM1 cells. The parallel development of the M+2 fraction in the 2 metabolites indicated that no loss of carbon occurred in the process by which methylsuccinyl-CoA was converted to propionyl-CoA.
Fig. 6.
Fig. 6.
Metabolic origin of glyoxylate in M. extorquens AM1 during growth on methanol. The contributions of central metabolic pathways to glyoxylate biosynthesis were calculated from the positional isotopomers of glycine measured by NMR. Results are expressed relative to methanol uptake, set arbitrarily to 1.0, and confidence intervals are given within brackets. TCA indicates tricarboxylic acid cycle; EMCP, ethylmalonyl-CoA pathway (23); and GRC, glyoxylate regeneration cycle (20).
Fig. 7.
Fig. 7.
Comparison of C1 assimilation pathways in isocitrate lyase-negative (ICL−; A), proposed from this study, and positive (ICL+; B) serine cycle methylotrophs. Note than CO2 is derived from methanol, and therefore the overall carbon balance is the same in the 2 organisms: 3 methanol→1 C3.
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