Characterization of the Mycobacterium tuberculosis H37Rv alkyl hydroperoxidase AhpC points to the importance of ionic interactions in oligomerization and activity

2001 ◽  
Vol 354 (1) ◽  
pp. 209-215 ◽  
Author(s):  
Radha CHAUHAN ◽  
Shekhar C. MANDE
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Enteric Bacteria ◽  
Enzymic Activity ◽  
Ionic Interactions ◽  
Three Dimensional Model ◽  
Mycobacterium Tuberculosis H37rv ◽  
Resistant Strains ◽  
Alkyl Hydroperoxidase

An alkyl hydroperoxidase (AhpC) has been found frequently to be overexpressed in isoniazid-resistant strains of Mycobacterium tuberculosis. These strains have an inactivated katG gene encoding a catalase peroxidase, which might render mycobacteria susceptible to the toxic peroxide radicals, thus leading to the concomitant overexpression of the AhpC. Although the overexpressed AhpC in isoniazid-resistant strains of M. tuberculosis may not directly participate in isoniazid action, AhpC might still assist M. tuberculosis in combating oxidative damage in the absence of the catalase. Here we have attempted to characterize the AhpC protein biochemically and report its functional and oligomerization properties. The alkyl hydroperoxidase of M. tuberculosis is unique in many ways compared with its well-characterized homologues from enteric bacteria. We show that AhpC is a decameric protein, composed of five identical dimers held together by ionic interactions. Dimerization of individual subunits takes place through an intersubunit disulphide linkage. The ionic interactions play a significant role in enzymic activity of the AhpC protein. The UV absorption spectrum and three-dimensional model of AhpC suggest that interesting conformational changes may take place during oxidation and reduction of the intersubunit disulphide linkage. In the absence of the partner AhpF subunit in M. tuberculosis, the mycobacterial AhpC might use small-molecule reagents, such as mycothiol, for completing its enzymic cycle.

scholarly journals Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

1976 ◽  
Vol 155 (3) ◽  
pp. 523-534 ◽  
Author(s):  
G Allen ◽  
J I Harris
Keyword(s):  
Conformational Changes ◽  
Bacillus Stearothermophilus ◽  
Three Dimensional ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Three Dimensional Model ◽  
Muscle Enzyme ◽  
Tyrosine Residues ◽  
Pig Muscle

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

scholarly journals Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

2021 ◽  
Vol 22 (7) ◽  
pp. 3731
Author(s):  
Andrea Kunova ◽  
Luca Palazzolo ◽  
Fabio Forlani ◽  
Giorgia Catinella ◽  
Loana Musso ◽  
...  
Keyword(s):  
Rice Blast ◽  
Rational Design ◽  
Structural Investigation ◽  
Resistance Mechanism ◽  
Three Dimensional ◽  
Pyricularia Oryzae ◽  
Cytochrome Bc1 ◽  
Cytochrome Bc1 Complex ◽  
Three Dimensional Model ◽  
Resistant Strains

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.

scholarly journals Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

2008 ◽  
Vol 52 (8) ◽  
pp. 2909-2914 ◽  
Author(s):  
Stéphanie Matrat ◽  
Alexandra Aubry ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Primary Structure ◽  
Three Dimensional ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Intrinsic Resistance ◽  
E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

scholarly journals Three-Dimensional Structures of Apo- and Holo-l-Alanine Dehydrogenase from Mycobacterium tuberculosis Reveal Conformational Changes upon Coenzyme Binding

2008 ◽  
Vol 377 (4) ◽  
pp. 1161-1173 ◽  
Author(s):  
Daniel Ågren ◽  
Matthias Stehr ◽  
Catrine L. Berthold ◽  
Shobhna Kapoor ◽  
Wulf Oehlmann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Coenzyme Binding ◽  
Alanine Dehydrogenase

scholarly journals Selectively Receptor-Blind Measles Viruses: Identification of Residues Necessary for SLAM- or CD46-Induced Fusion and Their Localization on a New Hemagglutinin Structural Model

2004 ◽  
Vol 78 (1) ◽  
pp. 302-313 ◽  
Author(s):  
Sompong Vongpunsawad ◽  
Numan Oezgun ◽  
Werner Braun ◽  
Roberto Cattaneo
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Measles Virus ◽  
Structural Model ◽  
Three Dimensional ◽  
Lymphocyte Activation ◽  
Cell Receptor ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
H Protein

ABSTRACT Measles virus (MV) enters cells either through the signaling lymphocyte activation molecule SLAM (CD150) expressed only in immune cells or through the ubiquitously expressed regulator of complement activation, CD46. To identify residues on the attachment protein hemagglutinin (H) essential for fusion support through either receptor, we devised a strategy based on analysis of morbillivirus H-protein sequences, iterative cycles of mutant protein production followed by receptor-based functional assays, and a novel MV H three-dimensional model. This model uses the Newcastle disease virus hemagglutinin-neuraminidase protein structure as a template. We identified seven amino acids important for SLAM- and nine for CD46 (Vero cell receptor)-induced fusion. The MV H three-dimensional model suggests (i) that SLAM- and CD46-relevant residues are located in contiguous areas in propeller β-sheets 5 and 4, respectively; (ii) that two clusters of SLAM-relevant residues exist and that they are accessible for receptor contact; and (iii) that several CD46-relevant amino acids may be shielded from direct receptor contacts. It appears likely that certain residues support receptor-specific H-protein conformational changes. To verify the importance of the H residues identified with the cell-cell fusion assays for virus entry into cells, we transferred the relevant mutations into genomic MV cDNAs. Indeed, we were able to recover recombinant viruses, and we showed that these replicate selectively in cells expressing SLAM or CD46. Selectively receptor-blind viruses will be used to study MV pathogenesis and may have applications for the production of novel vaccines and therapeutics.

Study of the structure–activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis

2001 ◽  
Vol 353 (3) ◽  
pp. 453-458 ◽  
Author(s):  
Nadine LEMAITRE ◽  
Isabelle CALLEBAUT ◽  
Frédéric FRENOIS ◽  
Vincent JARLIER ◽  
Wladimir SOUGAKOFF
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Cysteine Residue ◽  
Single Amino Acid ◽  
Three Dimensional Model ◽  
Mutant Proteins ◽  
Hydrophobic Cluster Analysis ◽  
Structural Modifications ◽  
Level Of Activity ◽  
Α Helix

In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (⩽ 0.004unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06–0.25unit/mg) and one (T167L) exhibited a wild-type activity (1.51units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 Å sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an α-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.

In Silico Analysis of S315T and S315R Mutations of Multidrug-resistant Mycobacterium tuberculosis Clinical Isolates from Karachi, Pakistan

2020 ◽  
Vol 13 (9) ◽  
Author(s):  
Muhammad Mumtaz Khan ◽  
Maria S Alves ◽  
Sadia Alam ◽  
Mohammad H Khan ◽  
Muhammad Jahangir ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistance ◽  
Conformational Changes ◽  
Gene Mutations ◽  
In Silico Analysis ◽  
Multidrug Resistant ◽  
Electrostatic Energy ◽  
Katg Gene ◽  
Resistant Strains ◽  
Binding Residues

Background: Tuberculosis is one of the most frequent and persistent global diseases causing millions of deaths every year. Pakistan lies at number 6 among the 22 most dominant countries, with multidrug resistance up to 15%. Isoniazid-resistant strains of Mycobacterium tuberculosis are gradually rising and seem to be more prevalent in developing countries. Mutations in the katG gene are considered to be responsible for the accusation of isoniazid resistance in M. tuberculosis. Objectives: The current study was designed to investigate the structural and functional associations of KatG gene mutations (S315R and S315T) and multidrug resistance in M. tuberculosis isolates from Karachi, Pakistan. Results: The present study revealed conformational changes in the structure of the KatG enzyme due to observed mutations, which led to induced alterations in isoniazid binding residues at the active site of the KatG enzyme. Furthermore, substantial changes were observed in interaction energy, ligand-receptor energy, electrostatic energy, salvation energy, and ligand-receptor conformational entropy. All these resultant modifications due to S315R and S315T mutations ultimately reduced the flexibility and stability of proteins at isoniazid-binding residues. Conclusions: This deviation in the consistency of protein texture eventually compromises the enzyme activity. It is well expected that the outcomes of the current study would provide a better understanding of the consequences of these mutations and provide a detailed insight into some previously unknown features.

scholarly journals Glucosamine-6-Phosphate Synthase de Mycobacterium tuberculosis um estudo in silico para predição de um modelo tridimensional refinado

2021 ◽  
Vol 7 (1) ◽  
pp. 1-9
Author(s):  
Wildrimak S. Pereira ◽  
Jhonatan M. S. Costa ◽  
Fábio L.C. Costa Júnior ◽  
Rômulo O. Barros ◽  
Ricardo M. Ramos
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
World Health ◽  
Dimensional Structure ◽  
Refined Model ◽  
Three Dimensional Model ◽  
Protein Database ◽  
Organization Studies ◽  
The World ◽  
Phosphate Synthase

Tuberculosis is one of the main causes of death by an infectious agent in the world, according to the World Health Organization. Studies indicate that enzymes involved in the biosynthesis of uridine diphospho-N-acetylglucosamine are essential for the life cycle of the bacterium. One of these enzymes is glucosamine-6-phosphate synthase (GlmS), which does not have a three-dimensional structure available in the protein database on the internet. In this work, structural bioinformatics methods (comparative modeling and molecular refinement) were used to build a refined three-dimensional model for the GlmS enzyme of Mycobacterium tuberculosis. The model was generated using four templates structures (crystallographic). The results obtained for the stereochemical and general parameters of the refined model were better than the original model and similarto those templates structures, validating the refined model.

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