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J. Biol. Chem., Vol. 275, Issue 46, 35932-35941, November 17, 2000
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From the Institut für Biotechnologie 1, Forschungszentrum
Jülich GmbH, 52425 Jülich, Germany and
§ Institut für Mechanik und Regelungstechnik,
Universität-GH Siegen, 57068 Siegen, Germany
The C3-C4
metabolite interconversion at the anaplerotic node in many
microorganisms involves a complex set of reactions. C3 carboxylation 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 a
13C 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 and
13C-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.
In Vivo Quantification of Parallel and Bidirectional
Fluxes in the Anaplerosis of Corynebacterium
glutamicum*
,
*
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.
To whom correspondence should be addressed. Tel.: 49-2461-613969;
Fax: 49-2461-612710; E-mail: a.de.graaf@fz-juelich.de.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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