DOI: 10.1128/jb.180.22.5809-5814.1998. Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from <i>Mycobacterium smegmatis</i>

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

10.1128/jb.180.22.5809-5814.1998

Crossref journal-article

American Society for Microbiology

Journal of Bacteriology (235)

Abstract

ABSTRACT 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 μg/ml.

Bibliography

Boshoff, H. I. M., & Mizrahi, V. (1998). Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from Mycobacterium smegmatis. Journal of Bacteriology, 180(22), 5809â5814.

Authors 2

Helena I. M. Boshoff (first)
Valerie Mizrahi (additional)

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Dates

Type	When
Created	5 years, 8 months ago (Dec. 31, 2019, 11:29 a.m.)
Deposited	4 years, 1 month ago (July 29, 2021, 2:25 p.m.)
Indexed	1 year ago (Aug. 4, 2024, 6:41 a.m.)
Issued	26 years, 9 months ago (Nov. 15, 1998)
Published	26 years, 9 months ago (Nov. 15, 1998)
Published Print	26 years, 9 months ago (Nov. 15, 1998)

Funders 0

None

BibTeX

@article{Boshoff_1998, title={Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from Mycobacterium smegmatis}, volume={180}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.180.22.5809-5814.1998}, DOI={10.1128/jb.180.22.5809-5814.1998}, number={22}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Boshoff, Helena I. M. and Mizrahi, Valerie}, year={1998}, month=nov, pages={5809–5814} }

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