Revisiting the ¹³C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

Abstract

During the past decade, 13 C-labeling based metabolic flux analysis (MFA) has increasingly been used to understand the effect of genetic alterations [1,2], changes in external conditions [3,4] and different nutritional regimes [5,6] on the metabolism of micro-organisms. 13 C-labeling based MFA relies on the feeding of 13 C-labeled substrate to a biological system, allowing the labeled carbon atoms to distribute over the metabolic network, and subsequently measuring the 13 C-label distributions of intracellular and ⁄ or secreted The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C 2 ) and dihydroxyacetone moiety (C 3 ) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C 2 and C 3 fragment pools in the pentose phosphate pathway and can lead to different label distributions for 13 C-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published 13 C-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several microorganisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. 7It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the 13 C-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the halfreaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.Abbreviations C 2 , glycolaldehyde moiety; C 3 , dihydroxyacetone moiety; e4p, erythrose 4-phosphate; f6p, fructose 6-phosphate; fbp, fructose 1,6-bisphosphate 3;4;5 ; g1p, glucose 1-phosphate; g6p, glucose 6-phosphate; g3p, glyceraldehyde 3-phosphate; MFA, metabolic flux analysis; p5p, pentose pool consisting of ribulose 5-phosphate, ribose 5-phosphate and xylulose 5-phosphate; PPP, pentose phosphate pathway; r5p, ribose 5-phosphate; s7p, sedoheptulose 7-phosphate; SS res , sum of squared residuals; S 2 res , estimated error variance; TA, transaldolase; TK, transketolase; TPP, thiamine pyrophosphate; x5p, xylulose 5-phosphate.

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

“…Metabolic Flux Analysis-Metabolic fluxes were derived using whole isotopologue analysis (15,33). In short, the procedure uses the cumomer balances and cumomer to isotopologue mapping matrices introduced by Wiechert et al (34) to calculate the isotopologue distributions of metabolites in a pre-defined stoichiometric network model for a given flux-set.…”

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

“…Third, the steps catalyzed by transketolase (TK) and transaldolase (TA) in the nonoxidative steps of the PPP are represented in terms of half-reactions to allow for the underlying mechanism of the enzymes and their lack of specificity (Kruger and von Schaewen, 2003;Kleijn et al, 2005;Selivanov et al, 2005). Conventional stoichiometric models of the nonoxidative reactions (Fig.…”

“…3,C and E). Theoretical analysis has shown that this formulation is essential for a correct description of the redistribution of label and that it allows the investigation of parallel pathways supported by different TK isozymes (Kleijn et al, 2005). Accordingly, the halfreaction scheme is used here to maximize the possibility of resolving cytosolic and plastidic contributions to the PPP fluxes.…”

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

“…These enzymes form a modified pathway, termed the pentose phosphoketolase pathway, in lactic acid bacteria and bifidobacteria (7). This pathway is utilized in the heterofermentative degradation of pentoses and hexoses to the end products CO 2 , ethanol, acetate, and lactate (8). Xfp can convert X5P generated at the end of the oxidative phase of the pentose phosphate pathway to glyceraldehyde 3-phosphate, which can enter the glycolytic pathway, and acetyl phosphate, which Ack can convert to acetate to generate ATP.…”