Evidence for isoniazid-dependent free radical generation catalyzed by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T)

The antitubercular agent isoniazid can be activated by Mycobacterium tuberc ulosis KatG using either a peroxidase compound I/II or a superoxide-depende nt oxyferrous pathway. The identity of activated isoniazid is unknown, but it has been suggested that it may be a free radical intermediate. In this w ork, EPR spin trapping experiments detected isoniazid-derived radicals gene rated during KatG-mediated oxidation via the peroxidase compound I/II pathw ay. On the basis of hyperfine splitting patterns and oxygen dependence, the se radicals were identified as the acyl, acyl peroxo, and pyridyl radicals of isoniazid. Isoniazid-resistant KatG(S315T) produced the same radicals fo und with KatG, while the less potent antitubercular agent nicotinic acid hy drazide produced the corresponding nicotinyl radicals. The time course of r adical production was similar for KatG and KatG(S315T), while a lower stead y-state level of radicals was produced from nicotinic acid hydrazide. These results support an earlier finding that the peroxidase pathway does not co rrelate with isoniazid resistance conferred by KatG(S315T). Trace amounts o f radicals were detected via the superoxide-dependent pathway. The low leve l of isoniazid-derived radicals found in the superoxide-dependent pathway m ay be due to scavenging by superoxide.