Biochemical, Biophysical, and Mechanistic Insights on a Putative Oxidoreductase From Mycobacterium tuberculosis That...

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. The emergence of Mtb's multidrug-resistant and extremely drug-resistant strains has imposed a great challenge for TB treatment. Hence, there is always a demand to explore new targets that may be crucial for the survival and pathogenicity of the bacilli. Oxidoreductases are a class of enzymes that transfer electrons in various biological pathways and reactions, at the expense of cellular NADPH/NADH. Here, we analyzed oxidoreductases from the H37Rv proteome and identified two uncharacterized putative oxidoreductases, Rv1260 and Rv1714. These putative oxidoreductases showed conservation among pathogenic and opportunistic mycobacterial species and were predicted to be virulence factors essential for the pathogen's survival. The 3D structural model and amino acid sequence of one of the oxidoreductases, Rv1260, showed similarities with tetracycline destructase, a flavin-dependent monooxygenase. Thin-layer chromatography and UV-visible spectroscopic experiments confirmed the presence of the FAD molecule in a bound form with the recombinant protein. Fluorescence quenching studies demonstrated a comparatively better affinity of NADPH than NADH with the protein. The protein also displayed efficient binding with chlortetracycline. Molecular dynamics simulations were employed to gain insights into the substrate binding and conformational changes in the protein. Moreover, the importance of the substrate binding region, the C-terminal helix, and the FAD binding cavity, located near the isoalloxazine ring, was highlighted. Overall, the study provides biochemical, biophysical, and mechanistic insights into one of the putative Mtb oxidoreductases. Based on our data, we propose that this protein may perform monooxygenation functions under specific redox conditions and contribute to the redox processes in Mtb.