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 approximately 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 binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.
Wengenack
N L
NL
Section of Hematology Research, Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Todorovic
S
S
Yu
L
L
Rusnak
F
F
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins; D004352:Drug Resistance, Microbial / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007538:Isoniazid / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000378:metabolism*; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D012694:Serine / Q000235:genetics; D013053:Spectrophotometry; D013912:Threonine / Q000235:genetics
D001426:Bacterial Proteins; D013912:Threonine; D012694:Serine; D010544:Peroxidases; C076136:catalase HPI; D007538:Isoniazid