title: Detection of novel coupled mutations in the katG gene (His276Met, Gln295His and Ser315Thr) in a multidrug-resistant Mycobacterium tuberculosis strain from Chennai, India, and insight into the molecular mechanism of isoniazid resistance using structural bioinformatics approaches.
abstract: This study reports on the structural basis of drug resistance targeting the katG gene in a multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strain with two novel mutations (His276Met and Gln295His) in addition to the most commonly reported mutation (Ser315Thr). A structural bioinformatics approach was used to predict the structure of the mutant KatG enzyme (MT). Subsequent molecular dynamics and docking studies were performed to explain the mechanism of isoniazid (INH) resistance. The results show significant conformational changes in the structure of MT leading to a change in INH binding residues at the active site, with a significant increase in the inhibition constant (Ki) of 5.67 μm in the mutant KatG-isoniazid complex (MT-INH) compared with the wild-type KatG-isoniazid complex (WT-INH). In the case of molecular dynamics studies, root mean square deviation (RMSD) analysis of the protein backbone in simulated biological conditions revealed an unstable trajectory with higher deviations in MT throughout the simulation process (1 ns). Moreover, root mean square fluctuation (RMSF) analysis revealed an overall increase in residual fluctuations in MT compared with the wild-type KatG enzyme (WT), whilst the INH binding residues of MT showed a decreased fluctuation that can be observed as peak deviations. Hence, the present study suggests that His276Met, Gln295His and Ser315Thr mutations targeting the katG gene result in decreased stability and flexibility of the protein at INH binding residues leading to impaired enzyme function.
authors.affiliation: L&T Microbiology Research Centre, Vision Research Foundation, 18 College Road, Chennai 600006, India.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Ramasubban
authors.forename: Kulandai Lily
authors.initials: KL
authors.lastname: Therese
authors.forename: Umashankar
authors.initials: U
authors.lastname: Vetrivel
authors.forename: Muthukumaran
authors.initials: M
authors.lastname: Sivashanmugam
authors.forename: Parvathy
authors.initials: P
authors.lastname: Rajan
authors.forename: R
authors.initials: R
authors.lastname: Sridhar
authors.forename: Hajib N
authors.initials: HN
authors.lastname: Madhavan
authors.forename: N
authors.initials: N
authors.lastname: Meenakshi
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics*; D019295:Computational Biology; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D007194:India; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid