Insights into the mechanisms of pyrazinamide resistance of three pyrazinamidase mutants N11K, P69T and D126N.

In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts the therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibit the growth of latent Mtb even at low pH. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, pncA gene contributes 72 to 99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe2+ ion. Among the mutations, D126N allosterically disturbed the position of the Fe2+ ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA also cause significant structural deformations that leading to PZA resistance. This study provides useful information that mutations in other than active parts may also cause proteins folding and ligand displacement effect, altering the biological functions.