Correlation between Isoniazid Resistance and Superoxide Reactivity in Mycobacterium tuberculosis KatG

Abstract

Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG) of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples to NADH forming an isoniazid-NADH adduct that ultimately confers antitubercular activity. We have compared the catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs, (RM)KatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotype KatGs, (WTP)KatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited any correlation with isoniazid resistance. The yields of isoniazid-NADH adduct formed were determined to be 1-5, 4-12, and 20-70-fold greater for the (WTP)KatGs than the (RM)KatGs for the compound I, II, and III pathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumption measurements) that correlates with drug resistance. Stopped-flow UV-visible spectroscopic studies revealed that all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decay were accelerated 4-12-fold in the presence of isoniazid (vs absence) for the (WTP)KatGs but were unaffected by the drug for the (RM)KatGs. A mechanism for isoniazid resistance which accounts for the observed reactivity for each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathway may be the primary factor in determining overall isoniazid resistance by specific KatG mutants, with secondary contributions arising from the compound I and II pathways.

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

“…Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.…”

“…Despite the considerable evidence that a peroxidatic process is involved in IN⅐NAD synthesis, attempts to rationalize the reaction entirely in terms of the peroxidatic pathway generally produced models that were incomplete from the standpoint of electron balance (5,7,10) or that involved hypothetical intermediates not characterized in any other system (5, 6). For example, a common scheme shows the isonicotinyl radical, generated in a peroxidatic reaction, reacting with NAD ϩ to yield IN⅐NAD, when such a reaction would actually yield the IN⅐NAD ϩ ⅐ radical (Fig.…”

“…Peroxidases, including KatGs, can generate O 2 . from O 2 and an electron donor such as INH or NADH (11, 15, 19 -21), and O 2 has a role, as yet not fully defined, in INH activation (4,5,11,14,17). Finally, three reports have described IN⅐NAD synthesis in mixtures of KatG, INH and NAD ϩ with no oxidant other than O 2 , conditions that also give rise to O 2 .…”

Oxidative Activation of Thiacetazone by the Mycobacterium tuberculosis Flavin Monooxygenase EtaA and Human FMO1 and FMO3

“…It claims over two million lives worldwide each year and dwells, hidden, in as many as two billion people (1). It is estimated that between 2005 and 2020, one billion people will be newly infected, over 125 million people will get sick, and 30 million will die of TB if control is not further strengthened (2). The worsening situation has prompted the World Health Organization to declare TB a global public health crisis.…”

Functional Implication Guided by Structure-Based Study on Catalase – Peroxidase (KatG) from Haloarcula Marismortui

“…It was reported that additionally, phenyl oxygen (Oη) of Y218 and amide nitrogen (N) backbone of M244 in the covalent adduct have hydrogen (H)-bonds with the guanisyl N 1 and N 2 of Arg409 (R409), respectively (Yamada et al, 2002). The covalent adduct has been shown to be generated by autocatalytic process (Ghiladi et al, 2005a). Crystallographic analysis of KatG from Bulkhorderia pseudomallei (Bp) has indicated that the side-chain of R409 (corresponding to HmKatG numbering) shows conformational change depending on both of pH and the physicochemical state of the active site (Carpena et al, 2005 M244L] in BpKatG considerably decreased the catalase activity, whereas the peroxidase activity was even enhanced (Regelsberger et al, 2000;Jakopitsch et al, 2004;Singh et al, 2004 Y218F] in the M244-Y218-W95 covalent adducts induced complete loss of the catalase activity, whereas the peroxidase activity was highly enhanced in Sy KatG (Jakopitsch et al, 2003a;Jakopitsch,et al, 2003b).…”