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Updated: Sep 27, 2026

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
The Flexible Peripheral Segments Modulate the Catalytic Activity of the FAD-Containing Monooxygenase EthA from
Dafeng Liu1,2, Xieping Sun1, Feng Yu1
1College of Biological Sciences and Technology, Yili Normal University, Yining 835000, China.
Abstract:
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.
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