title: Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase.
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.
authors: Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú. patricia.sheen@upch.pe
authors: Patricia
authors: P
authors: Sheen
authors: Patricia
authors: P
authors: Ferrer
authors: Robert H
authors: RH
authors: Gilman
authors: Gina
authors: G
authors: Christiansen
authors: Paola
authors: P
authors: Moreno-Román
authors: Andrés H
authors: AH
authors: Gutiérrez
authors: Jun
authors: J
authors: Sotelo
authors: Wilfredo
authors: W
authors: Evangelista
authors: Patricia
authors: P
authors: Fuentes
authors: Daniel
authors: D
authors: Rueda
authors: Myra
authors: M
authors: Flores
authors: Paula
authors: P
authors: Olivera
authors: José
authors: J
authors: Solis
authors: Alessandro
authors: A
authors: Pesaresi
authors: Doriano
authors: D
authors: Lamba
authors: Mirko
authors: M
authors: Zimic
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013054:Spectrophotometry, Atomic
chemical_list: D008670:Metals; D000581:Amidohydrolases; C022490:pyrazinamide deamidase