Structural modeling and biochemical characterization of MSMEG_0748, a MoxR ATPase from Mycobacterium smegmatis

<i>Mycobacterium tuberculosis</i> persists in a latent state within host macrophages by adapting to hostile conditions such as hypoxia and oxidative stress. Central to this adaptation, the carbon monoxide dehydrogenase (CO-DH) gene cluster supports intracellular survival by metabolizing carbon monoxide and nitric oxide. Among the uncharacterized genes within this cluster<i>, msmeg_0748</i>, annotated as a MoxR-type AAA+ ATPase in <i>Mycobacterium smegmatis</i> MC² 155, is hypothesized to act as a chaperone in CO-DH maturation. To characterize this protein, we employed AlphaFold3 structure prediction, validated by sequence alignment, SEC-MALS, biochemical assays, and biophysical analyses. MSMEG_0748 was confirmed to form a characteristic hexameric structure in solution. The structural models displayed conserved features including Walker A and B motifs and the MoxR-ATPase specific Helix 2 Insert (H2I). Furthermore, ATP/NADH- enzyme coupled assays confirmed its intrinsic ATP hydrolysis activity, which was dependent on the conserved catalytic glutamate (E146) in the Walker B motif. Co-immunoprecipitation experiments subsequently demonstrated a direct interaction between MSMEG_0748 and the adjacent VWA domain-containing protein, MSMEG_0751. These results provide comprehensive structural and functional insights into MSMEG_0748 and suggest the MSMEG_0748-MSMEG_0751 complex functions as the MoxR-VWA chaperone system essential for metalloenzyme maturation in mycobacteria.