Inhibition of the
enzyme Mycobacterium tuberculosis InhA (enoyl-
acyl carrier protein reductase) due to formation of an isonicotinoyl-
NAD adduct (IN-
NAD) from
isoniazid (INH) and
nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for
tuberculosis infection. INH action against mycobacteria requires
catalase-
peroxidase (KatG) function, and IN-
NAD adduct formation is catalyzed in vitro by M.
tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent
drug-resistant strain bearing KatG[S315T]. In this report, we describe how
hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-
NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the
isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with
NADH and INH, in the presence of KatG even in the absence of added
peroxide, due to endogenous
peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this
enzyme's reduced affinity for INH binding to the resting
enzyme and the catalytically competent
enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant
enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant
enzyme.