Whole-Cell Study Unveils Critical Mechanistic Elements, Regulatory Elements, and Inhibitor Targets of the...

The nontuberculous mycobacterium (NTM) Mycobacterium abscessus (Mab) has emerged as a global health concern due to its high intrinsic resistance toward antibiotics. The search for anti-NTM inhibitors requires novel well-characterized targets. The cytochrome bd (cyt-bd) oxidase, which serves as an alternate terminal oxidase in mycobacteria, is a chemically validated drug target in Mycobacterium tuberculosis (Mtb). However, no genetic, biochemical, or structural studies have been described for the Mab enzyme. Successful targeting of the Mab cyt-bd oxidase requires an in-depth understanding of its mechanistic and regulatory elements. Here, we generated a homology model of Mab cyt-bd, including the alternate menaquinol-binding pocket, the predicted oxygen channel, the proposed redox modulation site (C266-C285), and the salt bridge pair, keeping the cysteine residues in proximity. A heterologous system was developed for whole-cell functional studies to characterize the impact of mutations in these critical domains on enzyme activity. Mutating W9, E98, F103, or E263 to alanine inhibited the enzyme totally, underscoring their importance in menaquinol binding, oxygen reduction, and/or redox modulation. The Mab cyt-bd C285A mutant displayed a reduction in oxygen consumption and ATP formation, a phenomenon also presented for the Mtb C285A mutant. In summary, this study presents the first structural and biochemical characterization of Mab cyt-bd oxidase, providing insights into the importance of mechanistic and regulatory elements of the Mab enzyme in a whole-cell setup, which will be of relevance for the design of anti-NTM and antituberculosis hit molecules targeting this oxidase.