Structure-Guided Discovery of Novel Alternative Substrates for Mycobacterial Baeyer–Villiger Monooxygenase, EthA Through Integrated Computational and Biophysical Characterization

The Mycobacterium tuberculosis enzyme EthA (Rv3854c) impacts the survival of mycobacterial cells by converting inactive precursors of second-line anti-tubercular drugs like ethionamide (ETH) to an active form. This potentially inhibits cell wall mycolic acid biosynthesis. An Alphafold2-computed structural model of EthA reflects that it is a Class B Baeyer–Villiger Monooxygenase (BVMO) with two Rossmann fold motifs. Motif 1 is a FAD-binding motif which spans His4-Trp45 and Motif 2 is an overlapping FAD/NADP that spans between Asp174 and Gln215. We identified 4-Amino-3-methylnaphthol as the top-ranked structural alternative substrate candidate from the DrugBank library and 4-(1H-1,3-benzodiazole-2-yl)quinoline from the Maybridge library. Steady-state kinetic studies on the NADPH-dependent oxidation of EthA—using its lead natural substrate ETH and 4-methoxyphenylacetone—revealed that 4-methoxyphenylacetone is a promising structural alternative substrate. The molecular interactions and stability of ligands like ETH, DB15664, JFD03535 and 4-MPA with EthA model were analyzed using molecular dynamic simulations carried out over a 500-nanosecond time scale. Furthermore, the biophysical characterization of EthA was carried out through Dynamic Light Scattering, Differential Scanning Calorimetry, Circular Dichroism, Fluorescence and Fourier-Transform Infrared Spectroscopic methods.