Laboratorio de Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, San Martín de Porres, Lima, Peru.
Tuberculosis (Edinb). 2009 Mar;89(2):109-13. doi: 10.1016/j.tube.2009.01.004. Epub 2009 Feb 26.
Resistance of Mycobacterium tuberculosis to pyrazinamide is associated with mutations in the pncA gene, which codes for pyrazinamidase. The association between the enzymatic activity of mutated pyrazinamidases and the level of pyrazinamide resistance remains poorly understood. Twelve M. tuberculosis clinical isolates resistant to pyrazinamide were selected based on Wayne activity and localization of pyrazinamidase mutation. Recombinant pyrazinamidases were expressed and tested for their kinetic parameters (activity, k(cat), K(m), and efficiency). Pyrazinamide resistance level was measured by Bactec-460TB and 7H9 culture. The linear correlation between the resistance level and the kinetic parameters of the corresponding mutated pyrazinamidase was calculated. The enzymatic activity and efficiency of the mutated pyrazinamidases varied with the site of mutation and ranged widely from low to high levels close to the corresponding of the wild type enzyme. The level of resistance was significantly associated with pyrazinamidase activity and efficiency, but only 27.3% of its statistical variability was explained. Although pyrazinamidase mutations are indeed associated with resistance, the loss of pyrazinamidase activity and efficiency as assessed in the recombinant mutated enzymes is not sufficient to explain a high variability of the level of pyrazinamide resistance, suggesting that complementary mechanisms for pyrazinamide resistance in M. tuberculosis with mutations in pncA are more important than currently thought.
结核分枝杆菌对吡嗪酰胺的耐药性与编码吡嗪酰胺酶的 pncA 基因突变有关。突变型吡嗪酰胺酶的酶活性与吡嗪酰胺耐药水平之间的关系仍知之甚少。根据 Wayne 活性和吡嗪酰胺酶突变的定位，选择了 12 株耐吡嗪酰胺的结核分枝杆菌临床分离株。表达了重组吡嗪酰胺酶，并测试了它们的动力学参数（活性、kcat、Km 和效率）。通过 Bactec-460TB 和 7H9 培养来测量吡嗪酰胺耐药水平。计算了相应突变型吡嗪酰胺酶的耐药水平与动力学参数之间的线性相关性。突变型吡嗪酰胺酶的酶活性和效率因突变部位而异，其范围从低到高，与野生型酶相当接近。耐药水平与吡嗪酰胺酶活性和效率显著相关，但仅解释了其统计变异性的 27.3%。尽管吡嗪酰胺酶突变确实与耐药性有关，但在重组突变酶中评估的吡嗪酰胺酶活性和效率的丧失不足以解释吡嗪酰胺耐药水平的高度变异性，这表明 pncA 基因突变的结核分枝杆菌中除了吡嗪酰胺酶以外，可能还有其他机制在发挥作用。
