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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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