title: Isoniazid activation defects in recombinant Mycobacterium tuberculosis catalase-peroxidase (KatG) mutants evident in InhA inhibitor production.
abstract: 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 (KatG(A110V), KatG(A139P), KatG(S315N), KatG(L619P), KatG(L634F), and KatG(D735A)) were purified and characterized with respect to their catalase-peroxidase activities (in terms of k(cat)/K(m)), 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 KatG(L634F), KatG(A139P), and KatG(D735A) variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatG(S315N) and KatG(L619P) variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn(2+) to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors.forename: Chih-Jen
authors.initials: CJ
authors.lastname: Wei
authors.forename: Benfang
authors.initials: B
authors.lastname: Lei
authors.forename: James M
authors.initials: JM
authors.lastname: Musser
authors.forename: Shiao-Chun
authors.initials: SC
authors.lastname: Tu
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D007538:Isoniazid / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis*; D009195:Peroxidase / Q000378:metabolism; D010544:Peroxidases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D008345:Manganese; D010088:Oxidoreductases; D010544:Peroxidases; C076136:catalase HPI; D009195:Peroxidase; C085257:InhA protein, Mycobacterium; D007538:Isoniazid