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Journal of Bacteriology, November 1998, p. 5809-5814, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from Mycobacterium smegmatis

Helena I. M. Boshoff and Valerie Mizrahi*

Molecular Biology Unit, South African Institute for Medical Research, and Department of Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa

Received 9 July 1998/Accepted 9 September 1998

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 Km 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 µg/ml.

* Corresponding author. Mailing address: Molecular Biology Unit, SAIMR, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 2711-4899370. Fax: 2711-4899001. E-mail: 075val{at}chiron.wits.ac.za.

Journal of Bacteriology, November 1998, p. 5809-5814, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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