Uncovering Tuberculosis Resistance Behind Changes in the PZase Enzyme
Tuberculosis (TB) remains one of the infectious diseases that is difficult to control, particularly due to the emergence of drug-resistant Mycobacterium tuberculosis. One of the important drugs used in TB therapy 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; therefore, changes or mutations in this gene are thought 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, whereas R2 was resistant at 150 μg/mL. Genetic analysis revealed that R1 harbored three pncA mutations resulting in the amino acid substitutions Cys14Arg, Arg140His, and Ser179Gly, while R2 carried 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 mass 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, whereas that of PZase-R2 was 1.500 mM⁻¹·min⁻¹. These values were lower than that of the normal PZase, which reached 2.443 mM⁻¹·min⁻¹.
The reductions 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. The resulting reduction in enzyme activity is thought to contribute to the ability of M. tuberculosis to survive exposure to pyrazinamide.
Nevertheless, because each isolate carries multiple mutations simultaneously, further research is required 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 elucidate how each amino acid substitution affects the enzyme’s structure and its interaction 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 the corresponding protein, affect enzyme activity, and ultimately contribute to the ability of bacteria to survive drug exposure. Understanding the relationship between genes, enzymes, and drug resistance is expected to provide a foundation for developing improved resistance detection methods and more effective TB treatment strategies.
Keywords: pyrazinamide resistance, pncA gene, mutant PZase, catalytic kinetics.
Paper Link:
Purkan Purkan, Alivia Nadila, Bilqis Aliifa Nabilah, R Merlyn Sujatha, Wiwin Retnowati, Pratiwi Pudjiastuti, and Sofijan Hadi. Kinetical Analysis of Mutant PZase Enzyme to Uncover the Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates. New Microbiologica, 2026, 49(2), 107–116.
https://newmicrobiologica.org/wp-content/uploads/2026/07/MICRO_2_2026_2.1_FULL_496N570_Purkan-107-116.pdf
Authors and Affiliations
Purkan Purkan¹, Alivia Nadila¹, Bilqis Aliifa Nabilah¹, R Merlyn Sujatha³, Wiwin Retnowati⁴, Pratiwi Pudjiastuti¹, and Sofijan Hadi¹
¹ Department of Chemistry, Faculty of Science and Technology, Airlangga University, Jl. Mulyorejo, Surabaya 60115, Indonesia.
² Research Group of Biochemical Engineering, Enzyme Biotechnology and Gene Cloning, Airlangga University, Jl. Mulyorejo, Surabaya 60115, Indonesia.
³ Department of Biochemistry, St. Peter’s Institute of Higher Education and Research, Avadi, Chennai, Tamil Nadu, India.
⁴ Microbiology Division, Faculty of Medicine, Airlangga University, Jl. Prof. Dr. Moestajab, Surabaya, Indonesia.
Corresponding author: purkan@fst.unair.ac.id
Original source: “Uncovering Tuberculosis Resistance Behind Changes in the PZase Enzyme”
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, whereas R2 was resistant at 150 μg/mL. Genetic analysis revealed that R1 harbored three pncA mutations resulting in the amino acid substitutions Cys14Arg, Arg140His, and Ser179Gly, while R2 carried 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 mass 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, whereas that of PZase-R2 was 1.500 mM⁻¹·min⁻¹. These values were lower than that of the normal PZase, which reached 2.443 mM⁻¹·min⁻¹.
The reductions 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. The resulting reduction in enzyme activity is thought to contribute to the ability of M. tuberculosis to survive exposure to pyrazinamide.
Nevertheless, because each isolate carries multiple mutations simultaneously, further research is required 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 elucidate how each amino acid substitution affects the enzyme’s structure and its interaction 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 the corresponding protein, affect enzyme activity, and ultimately contribute to the ability of bacteria to survive drug exposure. Understanding the relationship between genes, enzymes, and drug resistance is expected to provide a foundation for developing improved resistance detection methods and more effective TB treatment strategies.
Keywords: pyrazinamide resistance, pncA gene, mutant PZase, catalytic kinetics.
Paper Link:
Purkan Purkan, Alivia Nadila, Bilqis Aliifa Nabilah, R Merlyn Sujatha, Wiwin Retnowati, Pratiwi Pudjiastuti, and Sofijan Hadi. Kinetical Analysis of Mutant PZase Enzyme to Uncover the Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates. New Microbiologica, 2026, 49(2), 107–116.
https://newmicrobiologica.org/wp-content/uploads/2026/07/MICRO_2_2026_2.1_FULL_496N570_Purkan-107-116.pdf
Authors and Affiliations
Purkan Purkan¹, Alivia Nadila¹, Bilqis Aliifa Nabilah¹, R Merlyn Sujatha³, Wiwin Retnowati⁴, Pratiwi Pudjiastuti¹, and Sofijan Hadi¹
¹ Department of Chemistry, Faculty of Science and Technology, Airlangga University, Jl. Mulyorejo, Surabaya 60115, Indonesia.
² Research Group of Biochemical Engineering, Enzyme Biotechnology and Gene Cloning, Airlangga University, Jl. Mulyorejo, Surabaya 60115, Indonesia.
³ Department of Biochemistry, St. Peter’s Institute of Higher Education and Research, Avadi, Chennai, Tamil Nadu, India.
⁴ Microbiology Division, Faculty of Medicine, Airlangga University, Jl. Prof. Dr. Moestajab, Surabaya, Indonesia.
Corresponding author: purkan@fst.unair.ac.id
This article is republished from the Popular Scientific Articles section of Universitas Airlangga (UNAIR) and has been adapted for publication on the UNAIR Chemistry website.
Original source: “Uncovering Tuberculosis Resistance Behind Changes in the PZase Enzyme”