title: Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds.
abstract: Recombinant wild-pyrazinamidase from H37Rv Mycobacterium tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer.
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rueda
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Patricia
authors.initials: P
authors.lastname: Sheen
authors.affiliation: Department of International Health, Bloomberg School of Public Health, The Johns Hopkins University, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Carlos
authors.initials: C
authors.lastname: Bueno
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Marco
authors.initials: M
authors.lastname: Santos
authors.affiliation: Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Mexico.
authors.forename: Victoria
authors.initials: V
authors.lastname: Pando-Robles
authors.affiliation: Laboratorio Universitario de Proteómica, Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico, Mexico.
authors.forename: Cesar V
authors.initials: CV
authors.lastname: Batista
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru. Electronic address: mirko.zimic@upch.pe.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zimic
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel / Q000379:methods; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D009535:Nicotinamidase / Q000378:metabolism*
chemical_list: D004220:Disulfides; D000581:Amidohydrolases; C022490:pyrazinamide deamidase; D009535:Nicotinamidase
keywords: Dimer; Drug resistance; Multimer; Mycobacterium; Nicotinamidase; Nicotinamide; Pyrazinamidase; Pyrazinamide; Reducing stress; Tuberculosis