title: Crystal structure of the pyrazinamidase of Mycobacterium tuberculosis: insights into natural and acquired resistance to pyrazinamide.
abstract: Pyrazinamidase (PncA) activates the first-line antituberculous drug pyrazinamide into pyrazinoic acid. The crystal structure of the Mycobacterium tuberculosis PncA protein has been determined, showing significant differences in the substrate binding cavity when compared to the pyrazinamidases from Pyrococcus horikoshii and Acinetobacter baumanii. In M. tuberculosis, this region was found to hold a Fe(2+) ion coordinated by one aspartate and three histidines, one of them corresponding to His57 which is replaced by Asp in Mycobacterium bovis, a species naturally resistant to pyrazinamide. The binding cavity also contains a Cys138-Asp8-Lys96 motif evocating a cysteine-based catalytic mechanism. Mutants have been constructed and investigated by kinetic and thermal shift assays, highlighting the importance of protein folding and thermal stability in the pyrazinamidase activity.
authors.affiliation: Faculté de Médecine Pitié-Salpêtrière, UPMC Université Paris 6, ER 5, EA1541, Bactériologie-Hygiène, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Petrella
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gelus-Ziental
authors.forename: Arnaud
authors.initials: A
authors.lastname: Maudry
authors.forename: Caroline
authors.initials: C
authors.lastname: Laurans
authors.forename: Rachid
authors.initials: R
authors.lastname: Boudjelloul
authors.forename: Wladimir
authors.initials: W
authors.lastname: Sougakoff
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry*; D000596:Amino Acids; D000995:Antitubercular Agents / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D004795:Enzyme Stability; D006358:Hot Temperature; D007501:Iron; D008958:Models, Molecular; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011718:Pyrazinamide / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D000995:Antitubercular Agents; D011718:Pyrazinamide; D007501:Iron; D000581:Amidohydrolases; C022490:pyrazinamide deamidase