scholarly journals Analysis of mutations leading to para-aminosalicylic acid resistance in Mycobacterium tuberculosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bharati Pandey ◽  
Sonam Grover ◽  
Jagdeep Kaur ◽  
Abhinav Grover
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Acid Resistance ◽  
Principal Component ◽  
Md Simulations ◽  
Missense Mutations ◽  
Aminosalicylic Acid ◽  
Folate Pathway ◽  
Key Enzyme ◽  
Key Residues

Abstract Thymidylate synthase A (ThyA) is the key enzyme involved in the folate pathway in Mycobacterium tuberculosis. Mutation of key residues of ThyA enzyme which are involved in interaction with substrate 2′-deoxyuridine-5′-monophosphate (dUMP), cofactor 5,10-methylenetetrahydrofolate (MTHF), and catalytic site have caused para-aminosalicylic acid (PAS) resistance in TB patients. Focusing on R127L, L143P, C146R, L172P, A182P, and V261G mutations, including wild-type, we performed long molecular dynamics (MD) simulations in explicit solvent to investigate the molecular principles underlying PAS resistance due to missense mutations. We found that these mutations lead to (i) extensive changes in the dUMP and MTHF binding sites, (ii) weak interaction of ThyA enzyme with dUMP and MTHF by inducing conformational changes in the structure, (iii) loss of the hydrogen bond and other atomic interactions and (iv) enhanced movement of protein atoms indicated by principal component analysis (PCA). In this study, MD simulations framework has provided considerable insight into mutation induced conformational changes in the ThyA enzyme of Mycobacterium.

scholarly journals Molecular Genetics of para-Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosis

2009 ◽  
Vol 53 (5) ◽  
pp. 2100-2109 ◽  
Author(s):  
Vanessa Mathys ◽  
René Wintjens ◽  
Philippe Lefevre ◽  
Julie Bertout ◽  
Amit Singhal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Acid Resistance ◽  
Clinical Isolates ◽  
Alternative Mechanism ◽  
Aminosalicylic Acid ◽  
Coding Region ◽  
First Line ◽  
Marked Contrast ◽  
Dilution Assay ◽  
Spontaneous Mutants

ABSTRACT The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

scholarly journals Computational Investigation of Bisphosphate Inhibitors of 3-Deoxy-d-manno-octulosonate 8-phosphate Synthase

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2370 ◽  
Author(s):  
Jéssica de Oliveira Araújo ◽  
Alberto Monteiro dos Santos ◽  
Jerônimo Lameira ◽  
Cláudio Nahum Alves ◽  
Anderson Henrique Lima
Keyword(s):  
Active Sites ◽  
Antimicrobial Agents ◽  
Md Simulations ◽  
Hydroxyl Groups ◽  
Ligand Complex ◽  
Effective Interactions ◽  
Key Enzyme ◽  
The Many ◽  
Key Residues ◽  
Computational Molecular Modeling

The synthase, 3-deoxy-d-manno-octulosonate 8-phosphate (KDO8P), is a key enzyme for the lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria and a potential target for developing new antimicrobial agents. In this study, computational molecular modeling methods were used to determine the complete structure of the KDO8P synthase from Neisseria meningitidis and to investigate the molecular mechanism of its inhibition by three bisphosphate inhibitors: BPH1, BPH2, and BPH3. Our results showed that BPH1 presented a protein–ligand complex with the highest affinity, which is in agreement with experimental data. Furthermore, molecular dynamics (MD) simulations showed that BPH1 is more active due to the many effective interactions, most of which are derived from its phosphoenolpyruvate moiety. Conversely, BPH2 exhibited few hydrogen interactions during the MD simulations with key residues located at the active sites of the KDO8P synthase. In addition, we hydroxylated BPH2 to create the hypothetical molecule named BPH3, to investigate the influence of the hydroxyl groups on the affinity of the bisphosphate inhibitors toward the KDO8P synthase. Overall, we discuss the main interactions between the KDO8P synthase and the bisphosphate inhibitors that are potential starting points for the design of new molecules with significant antibiotic activities.

scholarly journals Binding Pocket Alterations in Dihydrofolate Synthase Confer Resistance topara-Aminosalicylic Acid in Clinical Isolates of Mycobacterium tuberculosis

2013 ◽  
Vol 58 (3) ◽  
pp. 1479-1487 ◽  
Author(s):  
Fei Zhao ◽  
Xu-De Wang ◽  
Luke N. Erber ◽  
Ming Luo ◽  
Ai-zhen Guo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Binding Pocket ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Missense Mutations ◽  
Aminosalicylic Acid ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Mechanistic Basis

ABSTRACTThe mechanistic basis for the resistance ofMycobacterium tuberculosistopara-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursorpara-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance inM. tuberculosisstrain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates ofM. tuberculosisandMycobacterium bovis. From a panel of 85 multidrug-resistantM. tuberculosisclinical isolates, 5 were found to harbor mutations in thefolCgene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy offolCfully restored PAS susceptibility infolCmutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics forM. tuberculosisclinical isolates and for further defining the mode of action of this important tuberculosis drug.

scholarly journals Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para-Aminosalicylic Acid Resistance

2010 ◽  
Vol 54 (11) ◽  
pp. 4794-4798 ◽  
Author(s):  
Silke Feuerriegel ◽  
Claudio Köser ◽  
Leona Trübe ◽  
John Archer ◽  
Sabine Rüsch Gerdes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latin American ◽  
Acid Resistance ◽  
Multidrug Resistant ◽  
Nucleotide Polymorphisms ◽  
Aminosalicylic Acid ◽  
Phenotypic Resistance ◽  
Clear Cut ◽  
Development Of Resistance ◽  
Phylogenetic Lineages

ABSTRACT Single nucleotide polymorphisms (SNPs) involved in the development of resistance represent powerful markers for the rapid detection of first- and second-line resistance in clinical Mycobacterium tuberculosis complex (MTBC) isolates. However, the association between particular mutations and phenotypic resistance is not always clear-cut, and phylogenetic SNPs have been misclassified as resistance markers in the past. In the present study, we investigated the utility of a specific polymorphism in thyA (Thr202Ala) as a marker for resistance to para-aminosalicyclic acid (PAS). Sixty-three PAS-susceptible MTBC strains comprising all major phylogenetic lineages, reference strain H37Rv, and 135 multidrug-resistant (MDR) strains from Germany (comprising 8 PAS-resistant isolates) were investigated for the presence of Thr202Ala. In both strain collections, the Thr202Ala SNP was found exclusively in strains of the Latin American Mediterranean (LAM) lineage irrespective of PAS resistance. Furthermore, PAS MICs (0.5 mg/liter) for selected LAM strains (all containing the SNP) and non-LAM strains (not containing the SNP), as well as the results of growth curve analyses performed in liquid 7H9 medium in the presence of increasing PAS concentrations (0 to 2.0 mg/liter), were identical. In conclusion, our data demonstrate that the Thr202Ala polymorphism in thyA is not a valid marker for PAS resistance but, instead, represents a phylogenetic marker for the LAM lineage of the M. tuberculosis complex. These findings challenge some of the previous understanding of PAS resistance and, as a consequence, warrant further in-depth investigations of the genetic variation in PAS-resistant clinical isolates and spontaneous mutants.

scholarly journals Energetics and dynamics of a light-driven sodium-pumping rhodopsin

2017 ◽  
Vol 114 (27) ◽  
pp. 7043-7048 ◽  
Author(s):  
Carl-Mikael Suomivuori ◽  
Ana P. Gamiz-Hernandez ◽  
Dage Sundholm ◽  
Ville R. I. Kaila
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Rational Design ◽  
Md Simulations ◽  
Energy Transduction ◽  
Quantum Molecular Dynamics ◽  
Ion Pumps ◽  
Ion Translocation ◽  
Extracellular Side ◽  
Key Residues

The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521:48–53]. To elucidate its molecular mechanism for Na+ pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na+ binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.

scholarly journals Genetic Determinants Involved inp-Aminosalicylic Acid Resistance in Clinical Isolates from Tuberculosis Patients in Northern China from 2006 to 2012

2014 ◽  
Vol 59 (2) ◽  
pp. 1320-1324 ◽  
Author(s):  
Xiaobing Zhang ◽  
Liguo Liu ◽  
Yan Zhang ◽  
Guangming Dai ◽  
Hairong Huang ◽  
...  
Keyword(s):  
Acid Resistance ◽  
Northern China ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Aminosalicylic Acid ◽  
Genetic Determinants ◽  
Systematic Analysis ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Folate Pathway

ABSTRACTp-Aminosalicylic acid (PAS) is an important compound for treating multidrug-resistant tuberculosis (TB). Previous studies showed thatthyAmutations are often related to PAS resistance in clinical isolates. We performed a systematic analysis of isolate genotypes and detected mutations in three folate pathway genes (folC,thyA, andribD) in 61.1% (127/208) of PAS-resistant isolates, including 11 double mutants. This result expands our knowledge about the distribution and frequency of mutations related to PAS resistance in mycobacterial clinical isolates.

scholarly journals Analysis of changes of cavity volumes in predefined directions of protein motions and cavity flexibility

2021 ◽  
Author(s):  
German P. Barletta ◽  
Matias Barletta ◽  
Tadeo E. Saldaño ◽  
Sebastian Alberti
Keyword(s):  
Conformational Changes ◽  
Normal Mode Analysis ◽  
Growth Factor Receptor ◽  
Principal Component ◽  
Md Simulations ◽  
Mode Analysis ◽  
It Use ◽  
Nmr Structures ◽  
Structural Distortions ◽  
Protein Fluctuations

Dynamics of protein cavities associated with protein fluctuations and conformational plasticity is essential for their biological function. NMR ensembles, Molecular Dynamics (MD) simulations combined with Principal Component Analysis (PCA), and Normal Mode Analysis (NMA) provide appropriate frameworks to explore functionally relevant protein dynamics and cavity changes relationships. Within this context, we have developed ANA (Analysis of Null Areas), an efficient method to calculate cavity volumes. ANA is based on a combination of algorithms that guarantees its robustness against numerical differentiations. This is a unique feature with respect to other methods. Herein, we test ANA as a biophysical and bioinformatic method to analyze different structural and dynamics properties of cavities. In order to address this task, we have developed an updated and improved version of ANA that expands it use to quantify changes in cavity features, like volume and flexibility, due to protein structural distortions performed on predefined biologically relevant directions, e.g, directions of largest contribution to protein fluctuations (PCA modes) obtained by MD simulations or ensembles of NMR structures, collective NMA modes or any other direction of motion associated with specific conformational changes. A web page has been developed and its facilities are explained in detail, making the software available to all users. Firstly, we show that ANA can be useful to explore gradual changes of cavity volume and flexibility associated with protein ligand binding. Secondly, we perform a comparison study of the extent of variability between protein backbone structural distortions, and changes in cavity volumes and flexibilities evaluated for an ensemble of NMR active and inactive conformers of the epidermal growth factor receptor (EGFR) structures. Finally, we compare changes in size and flexibility between sets of NMR structures for different homologous chains of dynein.

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