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

The sulfur mobilization (SUF) system is the exclusive Fe-S cluster biosynthesis pathway found in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). Within this pathway, the SufC protein functions as an ATPase within the SufBC2D scaffold, playing a critical role in Fe-S cluster biogenesis. SufC ATPases from Mycobacterial species remain to be characterized, motivating this study which focuses on the cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using various biochemical, biophysical, and computational methods.

Upon purification, both SufC proteins were found existing as monomers in solution, despite sharing conserved domains. However, they displayed differing ATP-dependent kinetic efficiencies, indicating differences in their local environment and catalytic behavior. Further investigation using CRISPR interference targeting MSMEG_3124 demonstrated a reduction in M. smegmatis growth, emphasizing the vital role of SufC in cellular viability.

Computational modeling and virtual screening led to the identification of three potential compounds, from which Z2911048515 and Z66741142 were chosen for experimental evaluation. Circular dichroism spectroscopy analysis revealed subtle differences in Rv1463's spectral features in the presence of Z2911048515 and Z66741142, while substantial spectral variations were noted for MSMEG_3124.

Notably, Z66741142 exhibited alterations in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds demonstrated only minimal inhibition of SufC ATPase activity, exhibiting unique modes of action and suggesting they could serve as initial chemical scaffolds for further optimization and testing as effective inhibitors.

This study sheds light on the functional diversity of mycobacterial SufC proteins across both pathogenic and non-pathogenic species, paving the way for future mechanistic and structure-guided research within the indispensable SUF pathway.