Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase

Abstract

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. , and Mg 2+ 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 Zn 2+ . However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.

A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance

“…It has been speculated that the metalloenzyme PZAse of M. tuberculosis coordinates divalent metal ions with the participation of a metallochaperone [26]. We found genes associated to PZA resistance that are related with metal transporting and appeared mutated only in PZA-resistant strains.…”

“…Metal ions are necessary for PZAse activity and have a direct effect in the PZA antibiotic mechanism [5, 25, 26]. It has been speculated that the metalloenzyme PZAse of M. tuberculosis coordinates divalent metal ions with the participation of a metallochaperone [26].…”

“…These proteins are members of Cation Transporter ATPase family. This family is specialized in ion transport (Mg +2 , Zn +2 , Co +2 and Ni +2 ) across membrane using ATP hydrolysis energy [26]. In particular, CtpI ( P  < 0.05) and CtpE ( P  < 0.10) (mutations in these genes appeared only in resistant strains) are involved in the cellular homeostasis of metal cations.…”

Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity

“…Our results show that low concentrations of Zn 2ϩ (10 to 80 M) are capable of optimally reactivating PZAse, which has never been shown before. It had previously been reported (38,40) that 1.5 mM Zn 2ϩ was the optimum concentration needed to regain PZAse activity. However, that study did not evaluate as many points in the range of 1 M to 2,000 M Zn 2ϩ as are reported here; the points that coincide with those in this study are consistent.…”

“…Metal ions are necessary for PZAse activity and have significant implications for PZA's antibiotic mechanism of action (29,32,(36)(37)(38)(39). M. tuberculosis PZAse is inactivated by the removal of divalent ions and can be differentially reactivated (i.e., enzyme activity can be restored) (38,40); Zhang et al (38) observed reactivation by Fe 2ϩ and Mn 2ϩ when analyzed by inductively coupled plasma-optical emission spectroscopy (ICP-OES), and more recently, Sheen et al (40) showed in vitro reactivation by Co 2ϩ , Mn 2ϩ , and Zn 2ϩ but not Fe 2ϩ , Fe 3ϩ , or Mg 2ϩ . In this study, atomic absorption and X-ray fluorescence assays showed Zn 2ϩ to be present in recombinant PZAse expressed in E. coli (PZAse-EC), and 300 times as much Zn 2ϩ as enzyme was required for PZAse reactivation.…”