Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Abstract

There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases.

The Tuberculosis Prodrug Isoniazid Bound to Activating Peroxidases

“…Only then can the fine molecular details of catalysis be unraveled. Here we report the first crystal structures of INH bound to Saccharomyces cerevisiae CcP and soybean ascorbate peroxidase (sAPX), both of which are known activators of INH (1). We establish that the INH occupies the ␦-meso edge of the heme, and we also show that in an active site mutant of sAPX (W41A) INH can bind directly to the heme iron to become an inhibitor.…”

“…KatGs are bifunctional heme enzymes that exhibit both catalase activity and broad spectrum peroxidatic activity comparable with monofunctional peroxidases (4,5). The peroxidatic activity involves the formation of an oxidized ferryl intermediate (Equation 1, Compound I) that is subsequently reduced by substrate. This reduction usually occurs in two successive single-electron transfer steps as follows (Equations 2 and 3) (where P ϭ peroxidase, HS ϭ substrate, S ⅐ ϭ 1-electron oxidized form of substrate).…”

Comparative study of catalase-peroxidases (KatGs)

“…There was also a report mentioning katG delelion from the chromosome of two strains with high level INHresistance isolates (MIC > 50 mg/ml) (Zhang et al, 1992 (Musser, 1995 Musser, 1998). As a result, Ser315Thr is one of the most characterized variants of MtKatG and has recently had its crystal structure determined (Zhao et al, 2006 (Pierattelli e[ al., 2004 …”

Mammalian Heme Peroxidases and Mycobacterium tuberculosis

“…Its primary function is of catalase activity. However, its role as a peroxidase is well established and its peroxidative activity is comparable with those of other mono-functional heme peroxidases (Metcalfe et al, 2008; Pierattelli et al, 2004). Sofar, crystal structure of only unliganded-MtCP has been determined (Bertrand et al, 2004).…”