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

Abstract

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

High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

“…1b and 2A). Interestingly, the binding for NAD ϩ and ATP found in BpKatG occurs in this LL1 loop (29), suggesting that it might be a binding site for the as yet unknown physiological peroxidase substrates. As nonsecreted KatGs present the longest LL1 loop insertions (supplemental Fig.…”

Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria

“…The active derivatives of isoniazid are not fully characterized, but the main form is believed to be an isoniazid-NAD adduct (93,270,445). Isoniazid-NAD forms from the interaction of INH and NAD ϩ in the presence of Mn 2ϩ and O 2 , which is catalyzed by the catalase-peroxidase KatG (445). Indeed, the lack of a suitable catalase-peroxidase to catalyze this process is the primary reason why most mycobacteria are not susceptible to isoniazid.…”

“…Isoniazid, or isonicotinic acid hydrazide (INH), is a prodrug that the target organism must modify to its active form. The active derivatives of isoniazid are not fully characterized, but the main form is believed to be an isoniazid-NAD adduct (93,270,445). Isoniazid-NAD forms from the interaction of INH and NAD ϩ in the presence of Mn 2ϩ and O 2 , which is catalyzed by the catalase-peroxidase KatG (445).…”

Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis

“…We thank Ekkehard Collatz for English editing. connecting the distal pocket to the outside of the protein [24]. In contrast to the binding site …”