Biochemical Insights into Glutamate decarboxylase from Mycobacterium tuberculosis

Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen which establishes a life-long infection by residing within macrophages and halting the phagosome-lysosome fusion. In response to IFN-γ, activated macrophages deliver the pathogen to the acidic compartment of the phagolysosome where pH fluctuates between 4.5-5.0. M. tuberculosis possesses a gene coding for a putative glutamate decarboxylase (MtGadB), the role of which could be to overcome the acidity of the phagolysosome or, as part of the GABA-shunt, to compensate for the lack of the α-ketoglutarate dehydrogenase activity in M. tuberculosis. Objectives: With the current study we investigated the biochemical properties of MtGadB to unveil its physiological role in M. tuberculosis. Methods: MtGadB (carrying an N-terminal His6-tag) was expressed and purified in Escherichia coli via affinity chromatography and its pH-dependent spectroscopic and enzymatic properties were investigated and compared with those of the extensively studied E. coli homologue (EcGadB). Results: We developed a successful protocol for the expression and purification of His6-MtGadB. We found that the His6-tag negatively influences the oligomeric structure and the enzymatic activity of MtGadB, in agreement with the structural role of the N-terminus in EcGadB. UV-Visible and fluorescence spectroscopic studies showed that MtGadB shares with the EcGadB counterpart many biochemical similarities. This is in line with the full conservation of GAD-signature residues observed in MtGadB. Given the possible involvement of MtGadB in the GABA-shunt, this work sets the basis for a better understanding of the M. tuberculosis GABA-shunt enzymes.