Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase |
| |
Authors: | 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 |
| |
Institution: | Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú. patricia.sheen@upch.pe |
| |
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 Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), 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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|