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Biochemical Characterization and Computational Identification of Mycobacterium tuberculosis Pyrazinamidase in Some Pyrazinamide-Resistant Isolates of Iran

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Abstract

Pyrazinamide (PZA) is one the first line anti-tuberculosis drugs that require activation by the pyrazinamidase (PZase). Most PZA-resistant Mycobacterium tuberculosis strains have mutations in the pncA gene which encoding PZase that result in the reduction or loss of the enzyme activity. Herein, we have examined how various mutations, which have been found from the PZA-resistant M. t uberculosis strains in Iran, modify the PZase activity. To elucidate the possible role of these mutations, namely A143T (MUT1), L151S (MUT2), A143T/T168A/E173K (MUT3), in the bioactivity of the enzyme, the PZase and mutant genes were cloned, functionally expressed and biochemically and computationally characterized. In comparison to the PZase enzyme, the enzymatic efficiency of mutant enzymes was decreased, with MUT2 indicating the largest enzymatic efficiency reduction. Homology models of mutants were constructed based on the PZase X-ray crystal structure. Molecular modeling and substrate docking revealed that the wild-type has much stronger binding affinity to PZA than the mutants whereas MUT2 has the weakest binding affinity. In addition, the molecular dynamics simulations and the essential dynamics results illustrated that the positions of the 51st to 71st residues were more dynamics in MUT2 as compared to the other atoms in PZase, MUT1 and MUT3 which could decrease the K m and k cat values of the enzymes.

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Abbreviations

PZA:

Pyrazinamide

PZase:

Pyrazinamidase

MUT1:

Mutation A143T

MUT2:

Mutation L151S

MUT3:

Mutation A143T/T168A/E173K

TB:

Tuberculosis

MDR-TB:

Multidrug-resistant tuberculosis

XDR-TB:

Extensively drug-resistant tuberculosis

POA:

Pyrazinoic acid

pncA :

Pyrazinamidase coding gene

RMSD:

Root mean square deviation

MD:

Molecular dynamics

SPC:

Simple point charge

PME:

Particle Mesh Ewald

RMSF:

Root-mean-square fluctuation

ED:

Essential dynamics

MSD:

Mean square displacements

OD:

Optical density

PDB:

Protein Data Bank

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Acknowledgments

We thank all of our colleagues for help and advice. The authors express their gratitude to Biotechnology lab of Agriculture Faculty, Azarbayjan Shahid Madani University.

Author information

Authors and Affiliations

  1. Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti University of Medical Sciences, P. O. Box 1985717443, Tehran, Iran

    Farahnoosh Doustdar

  2. Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, P. O. Box 1985717443, Tehran, Iran

    Farahnoosh Doustdar

  3. Department of Biology, Faculty of Science, Azarbaijan Shahid Madani University, P.O. Box 53714-161, Tabriz, Iran

    Mohammad Pazhang, Mehrnoosh Safarzadeh, Davod Rabiei & Nader Chaparzadeh

  4. Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, P.O. Box 14395-1561, Tehran, Iran

    Faramarz Mehrnejad & Mohammad Mir-Derikvand

  5. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Hanieh Falahati

Authors
  1. Farahnoosh Doustdar
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  2. Mohammad Pazhang
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Corresponding author

Correspondence to Faramarz Mehrnejad.

Appendix

Appendix

See Fig. 11.

Fig. 11
figure 11

The kinetic date (Lineweaver–Burk curves) of wild type pyrazinamidase and mutants

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Doustdar, F., Pazhang, M., Mehrnejad, F. et al. Biochemical Characterization and Computational Identification of Mycobacterium tuberculosis Pyrazinamidase in Some Pyrazinamide-Resistant Isolates of Iran. Protein J 34, 181–192 (2015). https://doi.org/10.1007/s10930-015-9610-5

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