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- Materials and methods
The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell–Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), d-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 µm for both enzymes.
Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 µm and 290 µm, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 µm in both strains.
The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.
Corynebacterium glutamicum has a high NADPH demand for the overproduction of amino acids such as lysine and isoleucine. We previously found that the main site of NADPH generation during amino-acid production is the oxidative part of the pentose phosphate pathway . Therefore the reaction catalysed by the glucose-6-phosphate (Glc6P) dehydrogenase (EC 188.8.131.52):
- Glc6P+NADP+ 6-phosphoglucono-δ-lactone+NADPH
and 6-phosphogluconate (6PG) dehydrogenases (EC 184.108.40.206):
- 6PG+NADP+ Rib5P+CO2+NADPH,
(Rib5P is ribulose 5-phosphate) were examined in detail in this study.
There is detailed information about the flux distribution at the branch point between glycolysis and the pentose phosphate pathway in C. glutamicum. NMR analysis of proteinogenic amino acids after 13C labelling of two isogenic strains with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases has shown that the flux distribution between these pathways alters by a factor of 3 when the cofactor specificity is changed . According to similar studies the pentose phosphate pathway flux is reduced more than twofold in the case of fructose-grown cells as compared to glucose-grown cells . As yet little information is available about the detailed regulation of the respective enzymes in C. glutamicum.
In some cases the pentose phosphate pathway flux is regulated by changes of specific activities of the Glc6P and 6PG dehydrogenases, as, for example when using gluconate instead of glucose as the carbon source . In other cases, regulation by metabolite concentrations must be responsible, as with fructose- and glucose-grown C. glutamicum where no differences in specific enzyme activities, but strongly altered contributions of the pentose phosphate pathway were found . This has been explained by an increased intracellular fructose 1,6-bisphosphate (Fru1,6P2) concentration, which has been found to inhibit the Glc6P and 6PG dehydrogenases of several organisms [5–7]. Key metabolites like energy or electron donors have also been shown to play an important role in directing the carbon flux through central metabolism. For the pentose phosphate pathway of Saccharomyces cerevisiae it was found that ATP and NADPH are co-metabolites for directing the flux through the pentose phosphate pathway . Another example is glycolysis in Escherichia coli, which is mainly regulated by the intracellular ATP level .
The available data on the C. glutamicum enzymes clearly do not allow any well-founded conclusion on regulation of the pentose phosphate pathway flux in this organism. Therefore the aim of this work was to examine the Glc6P and 6PG dehydrogenases of C. glutamicum with respect to reaction mechanism and regulation by intracellular metabolites, and to correlate their in vivo activities with intracellular metabolite concentrations. In two isogenic C. glutamicum strains with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases, the in vivo pentose phosphate pathway fluxes based on pool sizes and enzyme kinetic constants were compared with fluxes measured by 13C labelling and NMR.