Superoxide reactivity of KatG: insights into isoniazid resistance pathways in TB

To gain insight into the mechanism of INH activation by KatG and to understand how resistance is conferred by the single active-site point mutation of KatG(S315T), we have employed pulse radiolysis as the means to initiate a catalytic pathway capable of mimicking the in vivo oxidation of isoniazid (INH). Radiolysis of a solution containing WT KatG revealed two intermediates: compound III (oxyferrous KatG) [415 (Soret), 545, 580 nm] formed [k1 = (4.47 +/- 0.91) x 105 M-1 s-1] in the absence of INH and compound II (410 (Soret), 540, 575 nm) formed [k1 = (4.43 +/- 0.69) x 105 M-1 s-1] in the presence of INH, with a comparison of the rates suggesting that compound III (rate-limiting) precedes compound II formation. By contrast, radiolysis of KatG(S315T) only led to compound III formation, whether INH was present [k1 = (4.72 +/- 0.99) x 105 M-1 s-1] or not [k1 = (4.51 +/- 1.38) x 105 M-1 s-1]. HPLC studies to determine the rates of INH-NADH adduct formation (an inhibitor of InhA) as catalyzed by KatG were also performed employing various oxidants: air [WT: (7.18 +/- 1.25) x 10-4, S315T: (0.74 +/- 0.39) x 10-4], superoxide (SOTS-1) [WT: (9.22 +/- 1.10) x 10-4, S315T: not detected], and tert-butylhydroperoxide [WT: (20.5 +/- 1.13) x 10-4, S315T: (10.15 +/- 0.19) x 10-4]. Taken together, the results from the pulse radiolysis work as well as the InhA inhibitor studies allow us to propose a mechanism capable of correlating the inability for the oxyferrous intermediate of KatG(S315T) to oxidize ("activate") INH to the suppressed formation of the INH-NADH adduct, thereby leading to INH resistance in Mycobacterium tuberculosis.