Regulation of central carbon metabolism in mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one third of the world's population and results in the death 1.5 million people a year. It is believed that M tuberculosis requires a high degree of metabolic plasticity to adapt to the various host environments in which it is found. Exploring these pathways may give rise to new targets for antibiotics design.;Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A. ACS was the first enzyme in M tuberculosis shown to be regulated via posttranslational acetylation by the cAMP--dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD+ and a mycobacterial sirtuin-like deacetylase. We provided evidence for the kinetic mechanism and the existence of the acetyl-adenylate intermediate via 31P-NMR spectroscopy. Furthermore, we dissected the regulatory role of K617 acetylation and showed that acetylation inhibits only the first adenylation half-reaction while leaving the second half-reaction intact. Finally, we demonstrated that the chemical mechanism of the enzyme relies on a conformational change between the two half reactions which is controlled by the protonation state of aspartate 525.;The M tuberculosis genome encodes a single pyruvate kinase gene, pykA, which is translated into an active pyruvate kinase enzyme. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and therefore can regulate metabolic flux through glycolysis. In this study, we show that pykA encodes an active pyruvate kinase and that this enzyme is subjected to allosteric activation by glucose-6-phosphate and AMP. We generated a DeltapykA M tuberculosis strain and show that pyruvate kinase is essential for glucose metabolism and co-metabolism. In the absence of pyruvate kinase, the knock-out strain grows as well as the WT on acetate and pyruvate. However, DeltapykA failed to grow on glucose and short chain fatty acids. The lack of growth is correlated with loss in viability and elevated levels of triose phosphates, pentose phosphates, methyiglyoxal and inhibition of ICDH. This study reveals that pyruvate kinase is essential for the metabolism of glucose and fatty acids.