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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+, Fe2+, 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.
Financial support: This research was funded by National Institute of Allergy and Infectious Diseases, National Institutes of Health United States Award 1R01TW008669-01, Third World Academy of Science (TWAS) Grant 08-070RG/BIO/LA-UNESCO FR:3240204464, Programme for Research and Training in Tropical Diseases (TDR)-World Health Organization Reference 2009/53662-0, and Fundación Instituto Hipólito Unanue. P.S. and M.Z. were supported by Tropical Medicine Research Centers (TMRC) New Tools to Understand and Control Endemic Parasites Grant 1 P01 AI51976 and Global Research Training Grant 3 D43 TW006581.
Authors' addresses: Patricia Sheen, Patricia Ferrer, Gina Christiansen, Paola Moreno-Román, Andrés H. Gutiérrez, Jun Sotelo, Wilfredo Evangelista, Patricia Fuentes, Daniel Rueda, Myra Flores, and Mirko Zimic, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, Lima, Perú, E-mails: email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, and email@example.com. Robert H. Gilman, Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, E-mail: firstname.lastname@example.org. Paula Olivera and José Solis, Instituto Peruano de Energía Nuclear, Lima, Perú, E-mails: polivera@IPEN.GOB.PE and email@example.com. Alessandro Pesaresi and Doriano Lamba, Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area Science Park—Basovizza, Trieste, Italy, E-mails: firstname.lastname@example.org and email@example.com.