Comparative Biochemical and Biophysical Insights into SufC ATPases from Mycobacterium tuberculosis and Mycolicibacterium smegmatis mc2155

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.