Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase

Patricia Sheen, Patricia Ferrer, Robert H. Gilman, Gina Christiansen, Paola Moreno-Román, Andrés H. Gutiérrez, Jun Sotelo, Wilfredo Evangelista, Patricia Fuentes, Daniel Rueda, Myra Flores, Paula Olivera, José Solis, Alessandro Pesaresi, Doriano Lamba, Mirko Zimic

Research output: Contribution to journalArticlepeer-review

Abstract

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co2+, Mn2+, and Zn2+ restored pyrazinamidase activity, only Co2+ enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu2+, Fe 2+, Fe3+, and Mg2+ did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn2+. However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.

Original languageEnglish (US)
Pages (from-to)153-161
Number of pages9
JournalAmerican Journal of Tropical Medicine and Hygiene
Volume87
Issue number1
DOIs
StatePublished - Jul 2012

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases
  • Virology

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