In Silico Analysis of S315T and S315R Mutations of Multidrug-resistant Mycobacterium tuberculosis Clinical Isolates from Karachi, Pakistan (original) (raw)
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International Journal of Antimicrobial Agents, 2011
This study reports on the structural basis of drug resistance targeting the katG gene in a multidrugresistant 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 m 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.
In Silico Analysis Of Isoniazid Resistance In Mycobacterium Tuberculosis
2014
Altered drug binding may be an important factor in isoniazid (INH) resistance, rather than major changes in the enzyme's activity as a catalase or peroxidase (KatG). The identification of structural or functional defects in the mutant KatGs responsible for INH resistance remains as an area to be explored. In this connection, the differences in the binding affinity between wild-type (WT) and mutants of KatG were investigated, through the generation of three mutants of KatG, Ser315Thr [S315T], Ser315Asn [S315N], Ser315Arg [S315R] and a WT [S315]) with the help of software-MODELLER. The mutants were docked with INH using the software-GOLD. The affinity is lower for WT than mutant, suggesting the tight binding of INH with the mutant protein compared to WT type. These models provide the <em>in silico</em> evidence for the binding interaction of KatG with INH and implicate the basis for rationalization of INH resistance in naturally occurring KatG mutant strains of <em&...
Mutation as a Molecular Determinant for Isoniazid Resistance in Mycobacterium tuberculosis
2017
Emergence of multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) is one of the reasons why tuberculosis (TB) continues to cause great mortality and morbidity in less-developed countries. The development of rapid diagnostic methods targeting genetic mutations associated with resistance to the anti-tuberculous drugs is essential to fight this deadly pathogen. Isoniazid (INH) has been included in the multidrug regimens for the treatment of drug-susceptible TB for the decades. In the worldwide setting, isoniazid resistance was highly prevalent and was observed in one of every seven TB cases. Since katG315 mutation is highly prevalent, the common mutation in the enzyme essential for the activation of the INH concerned with the mechanism of drug resistance and associated with high level resistance to INH, katG315 mutation was necessary to be identified by molecular method as a molecular determinant of INH resistant Mycobacterium tuberculosis. The...
Pneumonologia i alergologia polska, 2013
A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. A total of 43 (93%) strains contained mutations in the ka...
Antimicrobial agents …, 2010
Isoniazid (INH) is an effective first-line antituberculosis drug. KatG, a catalase-peroxidase, converts INH to an active form in Mycobacterium tuberculosis, and katG mutations are major causes of INH resistance. In the present study, we sequenced katG of 108 INH-resistant M. tuberculosis clinical isolates. Consequently, 9 novel KatG mutants with a single-amino-acid substitution were found. All of these mutants had significantly lower INH oxidase activities than the wild type, and each mutant showed various levels of activity. Isolates having mutations with relatively low activities showed high-level INH resistance. On the basis of our results and known mutations associated with INH resistance, we developed a new hybridization-based line probe assay for rapid detection of INH-resistant M. tuberculosis isolates.
The Ukrainian Biochemical Journal
Mutations in katg gene are often associated with isoniazid (INh) resistance in Mycobacterium tuberculosis strain. this research was perfomed to identify the katg mutation in clinical isolate (l8) that is resistant to INH at 1 μg/ml. In addition to characterize the catalase-peroxidase of KatG L8 and perform the ab initio structural study of the protein to get a more complete understanding in drug activation and the resistance mechanism. The katG gene was cloned and expressed in Escherichia coli, then followed by characterization of catalase-peroxidase of KatG. The structure modelling was performed to know a basis of alterations in enzyme activity. a substitution of a713g that correspond to asn238Ser replacement was found in the l8 katG. The Asn238Ser modification leads to a decline in the activity of catalase-peroxidase and INH oxidation of the L8 KatG protein. The catalytic efficiency (K cat /K M) of mutant katg asn238Ser respectively decreases to 41 and 52% for catalase and peroxidase. The mutant KatG asn238Ser also shows a decrease of 62% in INH oxidation if compared to a wild type katg (katgwt). the mutant asn238Ser might cause instability in the substrate binding site of katg, because of removal of a salt bridge connecting the amine group of asn238 to the carbo xyl group of Glu233, which presents in KatGwt. The lost of the salt bridge in the substrate binding site in mutant katg asn238Ser created changes unfavorable for enzyme activities, which in turn emerge as INh resistance in the l8 isolate of M. tuberculosis.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2011
The present study was carried out to investigate the presence of polymorphism in the N-acetyltransferase gene of 41 clinical isolates of Mycobacterium tuberculosis, that were resistant to isoniazid (INH) with no mutations in the hot spots of the genes previously described to be involved in INH resistance (katG, inhA and ahpC). We observed single nucleotide polymorphisms (SNPs) in ten of these, including the G619A SNP in five isolates and an additional four so far un-described mutations in another five isolates. Among the latter SNPs, two were synonymous (C276T, n = 1 and C375G, n = 3), while two more non-synonymous SNPs were composed of C373A (Leu→Met) and T503G (Met→Arg) were observed in respectively one and two isolates. Molecular modeling and structural analysis based in a constructed full length 3D models of wild type TBNAT (TBNAT H37Rv) and the isoforms (TBNAT L125M and TBNAT M168R) were also performed. The refined models show that, just as observed in human NATs, the carboxyl terminus extends deep within the folded enzyme, into close proximity to the buried catalytic triad. Analysis of tbnat that present non-synonymous mutations indicates that both substitutions are plausible to affect enzyme specificity or acetyl-CoA binding capacity. The results contribute to a better understanding of structure-function relationships of NATs. However, further investigation including INH-sensitive strains as a control group is needed to get better understanding of the possible role of these new mutations on tuberculosis control.
katG (SER 315 THR) Gene Mutation in isoniazid resistant Mycobacterium tuberculosis
Kathmandu University …, 2012
Isoniazid (INH) together with Rifampicin (RFP) forms the cornerstone of a short chemotherapy course for tuberculosis (TB) treatment. Mutation at codon 315 of katG gene is most prevalent in isoniazid resistant Mycobacterium tuberculosis (MTB) and is high in area with high TB incidence. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) has been found to be a reliable and effective tool for the identification of the specific gene alteration.
The Journal of Infectious Diseases, 2000
The prevalence of mutations at amino acid (aa) position 315 in the katG gene of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in The Netherlands and the mutation's association with the level of INH resistance, multidrug resistance, and transmission were determined. Of 4288 M. tuberculosis isolates with available laboratory results, 295 (7%) exhibited INH resistance. Of 148 aa 315 mutants, 89% had MICs of 5-10 mg/mL, whereas 75% of the other 130 INH-resistant strains had MICs of 0.5-1 mg/mL. Of the aa 315 mutants, 33% exhibited monodrug resistance, compared with 69% of other INH-resistant strains (P ! ). Multidrug resistance was found among 14% of the aa 315 mutants and 7% of the other .0001 INH-resistant strains ( ). The probability of being in an IS6110 DNA restriction frag-P 1 .05 ment length polymorphism cluster was similar for aa 315 mutants and INH-susceptible strains, but the probability was reduced in other INH-resistant strains. Thus, aa 315 mutants lead to secondary cases of tuberculosis as often as INH-susceptible strains do.