The drug pyrazinamide is a crucial component of existing treatments for tuberculosis – a disease that, in 2016, killed 1.7 million people. Due to its unique sterilising activity, inclusion of pyrazinamide in combination therapies reduces tuberculosis treatment from 12 to 6 months. The mechanism by which pyrazinamide kills Mycobacterium tuberculosis (the pathogen that causes tuberculosis), however, remains poorly understood. Recently, mutations in the gene coding for aspartate decarboxylase (ADC), a protein involved in pantothenate (vitamin B5) biosynthesis, have been shown to confer resistance to pyrazinamide’s activated derivative pyrazinoic acid. The mutated residues are primarily present in a C-terminal extension of ADC unique to Mycobacterium, and the molecular mechanisms governing resistance remain to be fully elucidated.
In this study, His-tagged M. tuberculosis ADC was expressed and purified from Escherichia coli and an enzyme-coupled assay was established to monitor the ADC’s decarboxylase activity in real time. Using this assay we have monitored the effect of pyrazinamide and pyrazinoic acid on ADC activity and are currently investigating the effect of resistance-conferring mutations. We have additionally studied the autocatalytic cleavage of M. tuberculosis ADC, which is required for its activation. We are investigating the possibility of the involvement of an accessory protein and the effect of pyrazinoic acid on cleavage. Our activity and cleavage data will be presented along with results from bioinformatic analyses. These were focused, in part, on exploring the recently proposed hypothesis that the C-terminus of M. tuberculosis ADC mediates a pyrazinoic acid-dependent interaction with the M. tuberculosis unfoldase ClpC1 and thereby its degradation.