Structural insights into sulfonamide degradation by a two-component flavin-dependent monooxygenase.

The sulfonamide-degrading monooxygenase sulX plays a dual role in bioremediation and antibiotic resistance, yet its molecular mechanism remains elusive. Here we report crystal structures of sulX in its ligand-free form, as an FMN-bound binary complex, and as ternary complexes with six distinct sulfonamides at resolutions ranging from 2.09 to 3.03 Å. These structures reveal that sulfonamides bind on the re face of the flavin isoalloxazine ring, with their conserved 4-aminophenol and sulfonyl moieties tightly anchored by π-stacking and hydrogen-bonding interactions, while the divergent aminated substituents extend into an open cleft with minimal specific contacts-a feature that explains the enzyme's remarkable capacity to accommodate diverse sulfonamide substrates. By integrating structural and biochemical data, we propose a catalytic mechanism involving F265-mediated substrate gating and H397-facilitated proton transfer. In addition, structure-based engineering yielded enhanced variants: A399S, which introduces new hydrogen bonds, showed a 37% increase in activity, and P422K achieved a 29% improvement. Our findings establish the structural basis of sulX-mediated sulfonamide degradation and provide a foundation for understanding sulfonamide resistance mechanisms.