Pyruvate kinase regulates the pentose-phosphate pathway in response to hypoxia in mycobacterium tuberculosis

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate-pyruvate-oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb.Ministry of Education (MOE)National Medical Research Council (NMRC)National Research Foundation (NRF)Accepted versionThis research was supported by the National Research Foundation of Singapore through the Singapore–MIT Alliance for Research and Technology Antimicrobial Resistance research program, and a Singapore–MIT Alliance for Research and Technology Postdoctoral Fellowship (W.Z.). During the course of this study, the J.L. laboratory was supported by grant NMRC/CBRG/ 0073/2014. The Y. M. laboratory was supported by the grant of MOE Tier 1 RG146/17 from Ministry of Education Singapore