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 Fe2+ 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.

In silico Analysis of Novel Mutation ala102pro Targeting pncA Gene of M. Tuberculosis

“…In M. tuberculosis, this region was found to hold a Fe 2+ ion coordinated by one aspartate and three histidines, the most crucial structural element in this loop appears to be the specific positioning of residue His57 which is directly involved in the coordination of the Fe 2+ ion. The overall architecture of the pyrazinamidase of M. tuberculosis is similar to that reported for the other pyrazinamidases of A. baumanii and P. horikoshii [11].…”

Wayne’s Assay: A Screening Method for Indirect Detection of Pyrazinamide Resistance in Mycobacterium Tuberculosis Clinical Isolates

“…Most of these strains were positive for PZase activity [11]. According to previous studies, mutations at certain amino acid sites, such as codons 8, 96, 134, and 138, at active sites, and codons 13, 49, 51, 57, and 68, coordinated to the Fe 2+ ion, are closely associated with MTB resistance to PZA [39,40]. Mutations at these sites can cause MTB to lose PZase activity and render the strain resistant to PZA.…”

Mammalian Heme Peroxidases and Mycobacterium tuberculosis

“…The crystal structure of Mycobacterium tuberculosis pyrazinamidase has been determined (Petrella et al, 2011) which shows that PncA folds into an /β single domain protein. It has an iron binding site involving residues Asp49, His51, His57 and His71 and consists of a catalytic triad with residues Cys138, Asp8 and Lys96.…”

“…Similarly, as revealed by the structure determination of the complex formed between LPO and PZA, PZA has been located in the substrate-binding site and interacts with substrate recognition residues of LPO (PDB ID: 3R4X) indicating a possible role of LPO in the conversion of PZA into an active form. Although the crystal structure of the PZA bound PncA is not known but a piece of information is available on the possible mode of ligand binding based on the molecular modeling data (Petrella et al, 2011). Therefore, it is of great interest that both prodrugs, INH and PZA bind to LPO specifically at the substrate-binding site on the distal heme side as the substrates bind to LPO (Singh et al, 2009) so that these compounds are converted into useful antimicrobial products.…”