Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis

Abstract

BackgroundMycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected.ResultsIn this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation.ConclusionsResults give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism.General significanceThis work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae

“…The enzyme was present primarily in an active dimeric form, but inactive octameric and higher oligomeric forms were also detected using size exclusion chromatography. Enzyme activity was not affected by any of the potential allosteric effectors described above, in contrast to enzymes such as ADP-glucose pyrophosphorylase from other actinomycetes (27, 33). …”

“…Potential allosteric regulators of OtsA were tested (fructose 6-phosphate, glucose 1-phosphate, mannose 1-phosphate, GTP, ATP, pyrophosphate, and orthophosphate), but none showed any effect on enzyme activity with GDP-glucose. This contrasts with the activation of the Mycobacterium tuberculosis enzyme by fructose 6-phosphate (27). Therefore, although able to use another purine diphosphoglucose donor, the enzyme had a preference for GDP-glucose and was not subject to allosteric regulation.…”

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice

“…However, we recently demonstrated that trehalose biosynthesis is mediated in mycobacteria only by the OtsA-OtsB2 and TreY-TreZ pathways [12], whereas TreS is involved in the conversion of trehalose to alpha-glucans and therefore consumes, rather than produces, this disaccharide [13]. In the OtsA-OtsB2 pathway, the trehalose-6-phosphate (T6P) synthase OtsA catalyzes the transfer of nucleoside diphosphate-activated glucose (ADP-glucose and, to a lesser extent, UDP-glucose [14]) to glucose-6-phosphate to yield T6P with the release of ADP/UDP. Subsequently, T6P phosphatase OtsB2 dephosphorylates T6P to trehalose.…”

α-Glucan biosynthesis and the GlgE pathway in Mycobacterium tuberculosis

“…Interestingly, the M. tuberculosis enzyme is activated by the glycolytic intermediates phosphoenol pyruvate (PEP) and glucose 6-phosphate (G6P; Figure 1) [11]. This would appear to increase flux from G1P through to ADPG and favour flux through to GlgA.…”

“…It had been assumed that the homologues of GlgA and GlgB in this organism are responsible for its synthesis. Indeed, the recombinant enzymes are capable of glycogen synthesis [10,11] and this is supported by genetic evidence [9].…”