S. Petersen et al., In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum, J BIOL CHEM, 275(46), 2000, pp. 35932-35941
The C-3-C-4 metabolite interconversion at the anaplerotic node in many micr
oorganisms involves a complex set of reactions. C-3, carboxylation to oxalo
acetate can originate from phosphoenolpyruvate and pyruvate, and at the Sam
e time multiple C-4-decarboxylating enzymes may be present. The functions o
f such parallel reactions are not yet fully understood. Using a C-13 NMR-ba
sed strategy, we here quantify the individual fluxes at the anaplerotic nod
e of Corynebacterium glutamicum, which is an example of a bacterium possess
ing multiple carboxylation and decarboxylation reactions. C. glutamicum was
grown with a C-13-labeled glucose isotopomer mixture as the main carbon so
urce and 13C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15
positional labels of biomass compounds were quantified. Applying a general
ly applicable mathematical model to include metabolite mass and carbon labe
ling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7%
to C-3 carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14
mmol/g dry weight/h exceeds the demand of carboxylated metabolites for bio
syntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate b
y phosphoenolpyruvate carboxykinase. This shows that the reactions at the a
naplerotic node might serve additional purposes other than only providing C
-4 metabolites for biosynthesis.