Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis
Fig 2
Mycobacterial GlgA is an M1P synthesizing glucosyltransferase.
(A) The preferred reaction catalyzed by GlgA from M. tuberculosis. (B) Recombinant M. tuberculosis GlgA converts ADP-glucose and G1P to M1P. 1H NMR spectroscopy was used to monitor the anomeric protons of ADP-glucose and G1P (1 mM each) together with signals associated with the product in the presence of GlgA. The spectra show the concomitant consumption of ADP-glucose (~5.5 ppm) and appearance of resonances consistent with the formation of α-1,4 glycosidic linkages (~5.32 ppm). Given the lack of any resonances associated with free reducing ends (~5.1 ppm) and the retention of those associated with an α-glucosyl phosphate residue (~5.36 ppm), these observations are consistent with the formation of M1P. (C) Mass spectrometry of the product of the M. tuberculosis GlgA reaction with ADP-glucose and G1P. The dominant ion in the spectrum is consistent with the presence of M1P (m/z 421.0749 observed with 421.0752 expected for [M-H]-). The next most abundant ion is consistent with the presence of the co-product, ADP (m/z 426.0218 observed with 426.0221 expected for [M-H]-). (D) The dependence of M. tuberculosis GlgA activity on ADP-glucose. GlgA activity was determined spectrophotometrically by monitoring the production of ADP. A representative triplicate dataset with 1 mM G1P is shown as means and SEM. The data conform to the Michaelis-Menten eq 1 (fit shown as the solid line giving r2 = 0.82). (E) The dependence of M. tuberculosis GlgA activity on G1P. GlgA activity was determined spectrophotometrically by monitoring the production of ADP. A representative triplicate dataset with 0.1 mM ADP-glucose is shown as means and SEM. Significant inhibition by G1P at concentrations >1 mM is apparent and the dataset conforms to a simple substrate inhibition eq 2 (fit shown as the solid line giving r2 = 0.99). (F) M. tuberculosis GlgA (Rv1212c) was heterologously expressed in the M. smegmatis ΔtreS(u) ΔglgA(u) c-glgE-tet-off mutant. Cells were cultivated for 24 h with or without 1 μg ml-1 ATc as indicated, and hot water extracts from 1 ml culture aliquots (normalized to OD600 nm = 0.5) were analyzed by TLC. Conditional silencing of the glgE gene results in M1P accumulation, demonstrating that M. tuberculosis GlgA synthesizes M1P in vivo.