Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase

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.