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

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.

Download Full-text

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

Biochemical Journal ◽

10.1042/bj1550523 ◽

1976 ◽

Vol 155 (3) ◽

pp. 523-534 ◽

Cited By ~ 3

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3540209 ◽

2001 ◽

Vol 354 (1) ◽

pp. 209-215 ◽

Cited By ~ 17

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.

Download Full-text

Substrate (aglycone) specificity of human cytosolic beta-glucosidase

Biochemical Journal ◽

10.1042/bj20021876 ◽

2003 ◽

Vol 373 (1) ◽

pp. 41-48 ◽

Cited By ~ 55

Author(s):

Jean-Guy BERRIN ◽

Mirjam CZJZEK ◽

Paul A. KROON ◽

W. Russell MCLAUCHLAN ◽

Antoine PUIGSERVER ◽

...

Keyword(s):

Structural Model ◽

Homology Modelling ◽

Three Dimensional ◽

Amino Acid Residues ◽

Dimensional Model ◽

Three Dimensional Model ◽

Mutant Proteins ◽

Metabolizing Enzyme ◽

Beta Glucosidase

Human cytosolic β-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays β-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (β/α)8-barrel fold characteristic of family 1 β-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val168→Tyr, Phe225→Ser, Tyr308→Ala and Tyr308→Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val168→Tyr mutation did not affect Km on p-nitrophenyl (pNP)-glycosides, but increased Km 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe225→Ser and Tyr308→Ala mutations, and, to a lesser degree, the Tyr308→Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

Download Full-text

Molecular Mechanisms of Mutations in Factor XIII A-subunit Deficiency: In vitro Expression in COS-cells Demonstrates Intracellular Degradation of the Mutant Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656113 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1068-1072 ◽

Cited By ~ 16

Author(s):

Aarno Palotie ◽

Hanna Mikkola ◽

Laszlo Muszbek ◽

Gizela Haramura ◽

Eija Hämäläinen ◽

...

Keyword(s):

Factor Xiii ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Subunit Gene ◽

Three Dimensional Model ◽

Mutant Proteins ◽

Cos Cells ◽

Intracellular Degradation ◽

A Subunit

SummaryFactor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four mis-sense mutations (Met242→Thr, Arg252→Ile, Arg326→Gln, Leu498 to Pro) and one stop mutation (Arg661→Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01380-07 ◽

2008 ◽

Vol 52 (8) ◽

pp. 2909-2914 ◽

Cited By ~ 13

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.

Download Full-text

A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPaseCauses Conditional Defects in Encapsidation and Uncoating

Journal of Virology ◽

10.1128/jvi.02877-15 ◽

2016 ◽

Vol 90 (14) ◽

pp. 6174-6186 ◽

Cited By ~ 17

Author(s):

Emmanuel Asare ◽

JoAnn Mugavero ◽

Ping Jiang ◽

Eckard Wimmer ◽

Aniko V. Paul

Keyword(s):

Nonstructural Protein ◽

Three Dimensional ◽

Virus Production ◽

Rna Replication ◽

Capsid Proteins ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Three Dimensional Model ◽

Cold Sensitive

ABSTRACTThe specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252of poliovirus 2CATPaseinteracts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPasehas important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus.In vivoprotein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase(K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection.IMPORTANCEEnterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPasepossessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2CATPasethat is involved in encapsidation but also identify a link between encapsidation and uncoating.

Download Full-text

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

Somma: Revista Cientifica do Instituto Federal de Educação, Ciência e Tecnologia do Piauí ◽

10.51361/somma.v7i1.49 ◽

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.

Download Full-text