Isoniazid Oxidation by Mycobacterium tuberculosis KatG:  A Role for Superoxide Which Correlates with Isoniazid Susceptibility

Wengenack, Nancy L.; Hoard, Heidi M.; Rusnak, Frank

doi:10.1021/ja992590a

Cited by 55 publications

(94 citation statements)

References 28 publications

(37 reference statements)

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“…Although activation (or at least oxidation) of INH by KatG has been initiated in vitro through various paths starting from resting KatG (44,45), only KatG Cmpd I (14) and the tyrosyl radical have been directly shown to be kinetically competent in reactions with this antibiotic. The results with small molecules presented here suggest that oxidations or, more likely, hydrogen atom abstractions may be initiated by the tyrosyl radical in a reaction separate from the heme-dependent peroxidase cycle in KatG.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

Chouchane

Girotto

et al. 2002

Journal of Biological Chemistry

100

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The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical ϳ0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for L-3,3-[ 2 H 2 ]tyrosine-labeled, but not L-2,4,5,6,7-[ 2 H 5 ]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

Chouchane

Girotto

et al. 2002

Journal of Biological Chemistry

100

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“…A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9,10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4,15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). 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.…”

Section: Isonicotinic Acid Hydrazide (Isoniazid or Inh)mentioning

confidence: 99%

“…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 .…”

Section: Isonicotinic Acid Hydrazide (Isoniazid or Inh)mentioning

confidence: 99%

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

Wiseman

Carpena

Feliz

et al. 2010

Journal of Biological Chemistry

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Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis Isonicotinic acid hydrazide (isoniazid or INH)3 is a widely used anti-tubercular pro-drug that requires activation in a reaction involving the catalase-peroxidase KatG of Mycobacterium tuberculosis (MtKatG) (1) whereby the hydrazine group is removed and the isonicotinyl portion is added to NAD ϩ to generate isonicotinyl-NAD or IN⅐NAD (see Fig. 1). Once formed, IN⅐NAD inhibits the synthesis of mycolic acids, and therefore, the growth of M. tuberculosis, by binding to the longchain enoyl acyl carrier protein reductase (InhA) (2). Despite understanding the role of IN⅐NAD in the inhibition of mycolic acid synthesis and knowing that KatG is required for INH activation in vivo, uncertainties about the mechanism of its formation remain.The involvement of MtKatG in INH activation suggested that the peroxidatic process had a role, and this provided a focus for several studies employing external oxidants such as peroxyacetic acid (3), t-butyl hydroperoxide (4 -6) and low levels of H 2 O 2 (7, 6) to activate the peroxidatic pathway for INH oxidation and activation. In addition, EPR studies have demonstrated that INH can serve as an electron source to reduce peroxidatic intermediates, including specific Trp ⅐ radical species following peroxyacetic acid oxidation of MtKatG (3,8).A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). 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. 1). The need to reduce this radical in an oxidizing environment suggests that more than a simple peroxidatic pathway is involved in the INH activation process. This rationale leads to superoxide, O 2. , a known reducing agent with

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“…The KatG protein and the KatG protein with the S315T mutation [KatG(S315T)] have comparable catalase and peroxidase activities, and KatG(S315T) is able to oxidize INH at equivalent rates using a hydroperoxide as oxidant, suggesting that the peroxidase activity of KatG may not be relevant for the in vivo activation of INH (31,44,45). In the presence of dioxygen but the absence of a hydroperoxide, INH is oxidized more slowly by KatG(S315T) than by KatG, a phenomenon that has been shown to involve superoxide and that may reflect why the S315T mutation confers INH resistance (44). Superoxide is formed during INH oxidation and is thought to be involved in the activation process (33)(34)(35).…”

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confidence: 99%

Oxidative Stress Increases Susceptibility of Mycobacterium tuberculosis to Isoniazid

Bulatovic¹,

Wengenack

Uhl

et al. 2002

Antimicrob Agents Chemother

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Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis.Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an Ϸ200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.Entering the 21st century, Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major cause of morbidity and mortality worldwide. TB causes 1.9 million deaths annually among a pool of infected individuals close to 2 billion people (47).Isoniazid (INH) is one of the most effective and widely used therapeutic agents for the treatment of TB. M. tuberculosis shows exceptional sensitivity to INH, being several orders of magnitude more sensitive than most other bacterial species. Much is known about the mechanism of INH activation, including that fact that it is a prodrug requiring activation by the catalase-peroxidase hemoprotein, KatG (13,18,19), in a process that requires molecular oxygen (49). It has been further shown that the activated form of INH forms a covalent adduct with NAD ϩ to generate a potent inhibitor of the InhA protein of M. smegmatis, an enoyl-acyl carrier protein reductase important in mycolic acid biosynthesis (26,30,40). Recent investigations have led to the hypothesis that the sensitivity of M. tuberculosis to INH may be governed by the level of expression of the alkyl hydroperoxide reductase, AhpC, since M. tuberculosis does not express the ahpC gene due to inactivation of the oxidative stress regulatory gene oxyR (5, 6, 32, 38, 52).Nevertheless, specific details regarding the mechanism of action of INH, such as the chemical nature of the activated form of INH, have yet to be determined. For example, InhA as the primary target for INH in M. tuberculosis has been questioned following the discovery of a covalent complex of INH, acyl carrier protein (AcpM), and the ␤-ketoacyl acyl carrier protein...

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Isoniazid Oxidation by Mycobacterium tuberculosis KatG: A Role for Superoxide Which Correlates with Isoniazid Susceptibility

Cited by 55 publications

References 28 publications

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

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

Oxidative Stress Increases Susceptibility of Mycobacterium tuberculosis to Isoniazid

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