Tuberculosis (TB) remains one of the infectious diseases that is difficult to control, particularly due to the emergence of drug-resistant Mycobacterium tuberculosis. One important drug used in TB treatment is pyrazinamide (PZA), which requires the enzyme pyrazinamidase (PZase) to convert it into its active form. The PZase enzyme is encoded by the pncA gene, meaning that changes or mutations in this gene are suspected to play an important role in the development of PZA resistance.
This study investigated the relationship between pncA gene mutations, PZase enzyme characteristics, and PZA resistance in two clinical isolates of M. tuberculosis, namely R1 and R2. Isolate R1 was resistant to PZA at a concentration of 100 μg/mL, while R2 was resistant at 150 μg/mL. Genetic analysis showed that R1 had three pncA mutations, resulting in the amino acid substitutions Cys14Arg, Arg140His, and Ser179Gly, whereas R2 had four mutations, resulting in the substitutions Ala26Ser, Ala38Pro, Thr135Pro, and Arg140His.
To determine the effects of these mutations on enzyme function, the pncA genes from both isolates were recombinantly expressed using Escherichia coli. The resulting mutant PZase proteins had a molecular weight of approximately 21 kDa and were subsequently analyzed for their catalytic activity. The results showed that the catalytic efficiency of PZase-R1 was 1.759 mM⁻¹·min⁻¹, while that of PZase-R2 was 1.500 mM⁻¹·min⁻¹. These values were lower than that of normal PZase, which reached 2.443 mM⁻¹·min⁻¹.
The decrease in catalytic efficiency reached 28.0% for PZase-R1 and 38.6% for PZase-R2. These findings indicate that mutations in pncA not only alter the genetic sequence but may also affect the ability of the PZase enzyme to perform its function. This reduction in enzyme activity is suspected to contribute to the ability of M. tuberculosis to survive exposure to pyrazinamide.
Nevertheless, since each isolate carries several mutations simultaneously, further research is still needed to determine the contribution of each individual mutation to changes in PZase function and PZA resistance. Approaches such as site-directed mutagenesis and protein structure modeling may help reveal how each amino acid substitution affects the enzyme’s structure and its interactions with the drug.
This study demonstrates that understanding TB resistance requires more than simply identifying mutations in DNA. Small changes in a gene can lead to changes in a protein, affect enzyme activity, and ultimately contribute to the ability of bacteria to survive exposure to drugs. Understanding the relationship between genes, enzymes, and resistance is expected to provide a foundation for developing resistance detection methods and more effective TB treatment strategies.
This article is a repost from the Popular Scientific Articles page of Universitas Airlangga (UNAIR) and has been adapted for publication on the UNAIR Chemistry website.
