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In Vivo Quantification of Parallel and Bidirectional Fluxes in the Anaplerosis of Corynebacterium glutamicum
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...
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Published in: | The Journal of biological chemistry 2000-11, Vol.275 (46), p.35932-35941 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M908728199 |