Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG (S315T)

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase−peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position ≈12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid ...