The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

Abstract

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.