Uncovering the structural impact of <scp>KatG</scp> Ser315 mutations in <i>Mycobacterium tuberculosis</i> via cryo‐ <scp>EM</scp>

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for a global health burden affecting over a quarter of the world's population. The increasing prevalence of drug-resistant TB poses a significant threat to current treatment strategies. Isoniazid (INH) is a first-line prodrug used in TB therapy, which requires activation by the catalase-peroxidase enzyme KatG. Upon activation, INH inhibits InhA, thereby disrupting mycolic acid biosynthesis, a crucial process for maintaining Mtb's distinctive, lipid-rich cell wall. The most common naturally occurring resistance-associated mutation in KatG is S315T, though other variants at this position, such as S315G, S315N, S315I, and S315R, have also been reported. In this study, we employ cryo-electron microscopy (cryo-EM) to investigate the structural basis of INH resistance conferred by these KatG variants. We present high-resolution cryo-EM structures that reveal heterogeneity in heme loading among the mutants. Detailed structural analysis highlights alterations in the hydrogen-bonding network and substrate access channel unique to each variant, offering direct comparisons with the wild-type (WT) KatG protein. Our findings provide a molecular explanation for clinical INH resistance and lay the groundwork for the rational design of next-generation anti-TB therapeutics.