The Flexible Peripheral Segments Modulate the Catalytic Activity of the FAD-Containing Monooxygenase EthA from Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health concern, particularly due to the emergence of drug-resistant strains. FAD-containing monooxygenase EthA activates the antitubercular prodrug ethionamide (ETH) in Mtb. However, the structural and functional mechanisms of Mtb EthA are not fully understood. Here, we report an AlphaFold2-predicted structural model of Mtb EthA, validated using Ramachandran analysis (91.7% residues in favored regions) and a ProSA Z-score of −10.96. Based on the results of molecular docking, site-directed mutagenesis was conducted. We found that R207 is required for EthA activity. Alanine substitutions at T186, S208, and T210 resulted in 5.8- to 7.2-fold reductions in activity. Conversely, deletion of three peripheral segments (residues 137–176, 315–336, and 419–460) enhanced activity by 1.9-, 1.5-, and 1.2-fold, respectively. These results provide a structural framework for EthA function and suggest that flexible peripheral regions may constrain catalytic activity, offering insights into ETH activation and potential mechanisms of drug resistance.