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 a/b 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 degrees 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. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

The multigene PE/PPE family is found exclusively in mycobacterium species. Rv3539, identified as PPE63, contains a conserved PPE domain at the N-terminal and a PE-PPE domain at the C-terminal. The study shows that Rv3539 has esterase activity, with the PE-PPE domain demonstrating higher enzyme activity. Additionally, the PPE domain is important for the thermostability and structural integrity of Rv3539.