Journal of Biological Chemistry
Volume 275, Issue 46, 17 November 2000, Pages 35932-35941
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METABOLISM AND BIOENERGETICS
In Vivo Quantification of Parallel and Bidirectional Fluxes in the Anaplerosis of Corynebacterium glutamicum *

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The C3-C4metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C3carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C4-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a13C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a 13C-labeled glucose isotopomer mixture as the main carbon source and13C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 ± 7% to C3 carboxylation. The total in vivo carboxylation rate of 1.28 ± 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C4 metabolites for biosynthesis.

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Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M908728199

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This work was supported by Degussa-Hüls AG.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.