Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable K-m values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 mu g/ml.