N-terminal PPE domain plays an integral role in extracellular transportation and stability of the immunomodulatory Rv3539 protein of the Mycobacterium tuberculosis.

Multigene PE/PPE family is exclusively present in mycobacterium species. Only few selected genes of this family have been characterized till date. Rv3539 was annotated as PPE63 with conserved PPE domain at N-terminal and PE-PPE at C-terminal. An α/β hydrolase structural fold, characteristic of lipase/esterase, was present in the PE-PPE domain. To assign the biochemical function to Rv3539, the corresponding gene was cloned in pET-32a (+) as full-length, PPE, and PE-PPE domains individually, followed by expression in E. Coli C41 (DE3). All three proteins demonstrated esterase activity. However, the enzyme activity in the N-terminal PPE domain was very low. The enzyme activity of Rv3539 and PE-PPE proteins was approximately same with the pNP-C4 as optimum substrate at 40 °C and pH 8.0. The loss of enzyme activity after mutating the predicted catalytic triad (Ser296Ala, Asp369Ala, and His395Ala) found only in the PE-PPE domain, confirmed the candidature of the bioinformatically predicted active site residue. The optimal activity and thermostability of the Rv3539 protein was altered by removing the PPE domain. CD-spectroscopy analysis confirmed the role of PPE domain to the thermostability of Rv3539 by maintaining the structural integrity at higher temperatures. The presence of the N-terminal PPE domain directed the Rv3539 protein to the cell membrane/wall and the extracellular compartment. The Rv3539 protein could generate humoral response in TB patients. Therefore, results demonstrated that Rv3539 demonstrated esterase activity. PE-PPE domain of Rv3539 is functionally automated, however, N-terminus domain played a role in protein stabilization and its transportation. Both domains participated in immunomodulation.