Cloning, expression, purification, and characterization of glutamate decarboxylase (Rv3432c) from Mycobacterium tuberculosis.

Abstract

Glutamate decarboxylase (Gad), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA), consuming a proton in the process and thereby contributing to intracellular pH homeostasis in bacteria. However, the presence and function of the Gad-dependent mechanism in mycobacteria remain largely unexplored. This study aimed to characterize Gad activity in Mycobacterium tuberculosis (Mtb). We detected Gad activity in live cells of both Mtb and Mycobacterium smegmatis (MS). Gad activity and GABA was also detected in cell lysates of Mtb and MS. The gadB gene from Mtb was cloned, expressed, and GadB protein was purified under native conditions using MS as an expression host. Initial attempts to express GadB in Escherichia coli (E. coli) resulted in the overexpressed protein being present in the insoluble fraction and was enzymatically inactive when purified under denaturing conditions. Subsequently, an acetamide-inducible expression system was employed in MS for successful overexpression and purification of recombinant GadB. 6 × His-GadB was purified using immobilized metal affinity chromatography, and its molecular weight was determined to be ~ 51.2 kDa by SDS-PAGE. The purified 6 × His-GadB enzyme was active at both neutral and acidic pH. Its activity was found to be PLP-dependent, with optimal activity at pH 7.2 and 50°C. These findings suggest that Gad is expressed in Mtb both in normal and in acidic medium, supporting the possible existence of a Gad-dependent acid resistance mechanism in Mtb.