Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation

Abstract

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

Kinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles

“…Tagatose-6-phosphate, another potential substrate of PfkA, supported by kinetic data of the PfkA belonging to M. tuberculosis (Table 4 ) [ 43 ], does raise the question if T6P is also a substrate of C. thermocellum PfkA and if there are conditions under which accumulation of T6P will trigger the expression of said PfkA. Such an assumption would be dependent on C. thermocellum having all the other required enzymes to produce T6P, which goes beyond the scope of this paper.…”

Prolonged survival of a patient with active MDR-TB HIV co-morbidity: Insights from a Mycobacterium tuberculosis strain with a unique genomic deletion

“…In turn, Rv2028 and Rv2034 positively regulate the expression of the dosR gene [30,32]. Many of the genes within Del-X are induced by the conditions typical of macrophages phagosomes, such as hypoxia, starvation, low levels of nitric oxide, and acidic pH [30,[33][34][35][36][37][38][39]. hspX actively contributes to slowing the growth of Mtb immediately following infection and is essential for its resistance to low oxygen stress [29,30].…”

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

“…The homolog of PfkB in M. tuberculosis is also upregulated during hypoxia, can catalyse the reverse gluconeogenic reaction (albeit with low efficiency), and exhibits lower activity, sensitivity to allosteric regulators and substrate specificity than PfkA, which is essential for growth on glucose 75,76 . We did not identify PfkA in our proteomics dataset, but it was not found to be differentially regulated in other microarray 34 and transcriptomic 77 datasets comparing hypoxic and aerated cultures of M. smegmatis .…”

“…Similarly, in M. tuberculosis, which lacks a PEPS homolog, the metabolic rerouting towards TAG synthesis plays a key role in growth arrest by depleting PEP 73 and acetyl-CoA 74 , and rerouting towards the PPP is mediated by pyruvate kinase 41 . We can only speculate about possible posttranscriptional regulation when the abundance of metabolites and associated enzymes do not correlate, though investigation of these mechanisms require intensive biochemical characterisation 75 .…”