Effect of Multiple Mutation in PZase From Clinical Isolate of Pyrazinamide-Resistant Mycobacterium tuberculosis on Its activity and structure

Mutations in the pncA gene, which encodes the enzyme PZase, are strongly linked to pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. The PZase R1 variant, carrying three amino acid substitutions (Cys14Arg, Arg140His, and Ser179Gly), is associated with resistance at PZA concentrations up to 100 μg/mL. However, its structural and functional characteristics remain poorly understood. This study examines the impact of these triple mutations on the structure and catalytic activity of PZase R1 compared to the wild type. Recombinant PZase was expressed in E. coli BL21 (DE3), producing a 22 kDa protein confirmed by SDS-PAGE. The R1 mutant showed a 31% reduction in enzyme activity. Structural modeling revealed a 0.27 Å deviation (RMSD) from the wild-type structure, indicating significant conformational change. These mutations disrupted local intramolecular interactions that contribute to the structural deviation. Specifically, the Cys14Arg substitution formed a new hydrogen bond with Asn11, while Arg140His and Ser179Gly resulted in the loss of electrostatic and hydrogen bonds, respectively, in the mutant protein. Molecular dynamic simulation showed that mutant PZase R1 had higher root mean square fluctuation (RMSF) and radius of gyration (RoG), indicating it has lower structural stability and compactness. Binding affinity analysis revealed weaker PZA binding in the mutant PZase R1 compared to the wild type. Overall, the combined structural damage and reduced activity due to the three mutations significantly contribute to PZA resistance in the Mycobacterium tuberculosis R1 isolate. Future studies using site-directed mutagenesis are needed to identify the specific residues most responsible for reduced PZase activity.