Background: Multi drug-resistant tuberculosis is a major health threat to humans.
Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed
mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was
up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated
to alanine frequently in the INH resistant strain of M. tuberculosis.
Methods: In this report, an array of computational approaches was used to understand the role of
Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability
and degree of INH modification was demonstrated using the molecular dynamics method. The
mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method.
Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed
in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by
agar plate culture spot and CFUs count assay.
Results: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the
substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap
region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site
cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD,
RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period
while the native protein displayed comparatively more fluctuations. Moreover, docking studies
showed that INH was bound into the active pocket of the mutant with considerable binding energy
(−6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50
ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular
bonding network with the neighbor amino acid residues. In vitro studies clearly suggested
that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared
to wild type.
Conclusion: Overall, this study at the outset suggested that the mutation observed in drug resistant
strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement,
leading to the possibility for its modification. In vitro results supported our in silico findings.