Biochemical characterization of Glutamate decarboxylase from Mycobacterium tuberculosis

During the infectious process Mycobacterium tuberculosis typically encounters an acid stress in the human macrophage phago(lyso)some (pH 4.5-6.0) [1]. Nonetheless, how M. tuberculosis survives this challenge is little understood. We have characterized M. tuberculosis glutamate decarboxylase (MtGadB). GadB catalyzes the decarboxylation of L-glutamate to yield GABA, while consuming one H+/catalytic cycle. It is a key enzyme in the potent glutamate-dependent acid resistance system found in many neutralophilic bacteria [2]. So far the only role proposed for MtGadB is to fill the interrupted TCA cycle in M. tuberculosis [3]. However, the perfect conservation of all key residues known as “GAD signature” [2] suggests that MtGadB may also play a role in protecting M. tuberculosis from acid stress. MtgadB was cloned into two different vectors, for expression with/without a His-tag. The best conditions for expression were screened by a rapid colorimetric assay. MtGadB was purified using either an ion exchange or an affinity chromatography. The oligomeric state was assessed by gel filtration chromatography. The pH-dependent activity and titration curves were compared with those of the E. coli and Brucella microti GadBs that we have characterized [4]. This study sets the basis to uncover the role of GadB in M. tuberculosis biology, and its potential as a “druggable” target. [1] Vandal et al. (2009) J Bacteriol. 191:4714-4721. [2] De Biase and Pennacchietti (2012) Mol. Microbiol 86:770-786. [3] Tian et al. (2005) PNAS 102:10670-10675. [4] Grassini et al. (2015) FEBS Open Bio 5:209-18.