title: PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase.
abstract: Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M-1s-1, respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×106M-1s-1at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106and 1×106M-1s-1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
authors.forename: Aníbal M
authors.initials: AM
authors.lastname: Reyes
authors.affiliation: Instituto de Química y Físicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB), Universidad de Buenos Aires and CONICET, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Diego S
authors.initials: DS
authors.lastname: Vazquez
authors.affiliation: Departamento de Química Inorgánica, Analítica y Química-Física and INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Ari
authors.initials: A
authors.lastname: Zeida
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
authors.forename: Martín
authors.initials: M
authors.lastname: Hugo
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Unidad de Biología Molecular-Institut Pasteur Montevideo, Montevideo, Uruguay.
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Piñeyro
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
authors.forename: María Inés
authors.initials: MI
authors.lastname: De Armas
authors.affiliation: Departamento de Química Inorgánica, Analítica y Química-Física and INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Darío
authors.initials: D
authors.lastname: Estrin
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.affiliation: Instituto de Química y Físicoquímica Biológicas "Prof. Alejandro C. Paladini" (IQUIFIB), Universidad de Buenos Aires and CONICET, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Javier
authors.initials: J
authors.lastname: Santos
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay. Electronic address: madiat@fmed.edu.uy.
authors.forename: Madia
authors.initials: M
authors.lastname: Trujillo
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D000445:Aldehyde Oxidoreductases; C027902:fatty acid reductase
keywords: Fatty acid hydroperoxides; Mycobacterium tuberculosis; Peroxidatic and resolving cysteine; Peroxiredoxin; Peroxynitrite; Thiol-dependent peroxidase; Thioredoxin