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Antimicrobial Agents and Chemotherapy, February 2003, p. 670-675, Vol. 47, No. 2
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.2.670-675.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Isoniazid Activation Defects in Recombinant Mycobacterium tuberculosis Catalase-Peroxidase (KatG) Mutants Evident in InhA Inhibitor Production

Chih-Jen Wei,1,{dagger} Benfang Lei,2 James M. Musser,2 and Shiao-Chun Tu1*

Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001,1 Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 598402

Received 29 April 2002/ Returned for modification 24 May 2002/ Accepted 12 November 2002

Mycobacterium tuberculosis KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical Mycobacterium tuberculosis isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatGA110V, KatGA139P, KatGS315N, KatGL619P, KatGL634F, and KatGD735A) were purified and characterized with respect to their catalase-peroxidase activities (in terms of kcat/Km), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatGL634F, KatGA139P, and KatGD735A variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatGS315N and KatGL619P variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn2+ to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.

* Corresponding author. Mailing address: Department of Biology and Biochemistry, 369 Science and Research 2, University of Houston, Houston, TX 77204-5001. Phone: (713) 743-8359. Fax: (713) 743-8351. dtu{at}uh.edu.

{dagger} Present address: Laboratory of Developmental Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019.

Antimicrobial Agents and Chemotherapy, February 2003, p. 670-675, Vol. 47, No. 2
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.2.670-675.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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