|dc.description.abstract||Sulfur-containing biomolecules participate in various chemical and structural functions in enzymes. When sulfate is limiting, bacteria upregulate ssi enzymes to utilize organosulfonates as an alternative source. The alkanesulfonate monooxygenase enzymes mitigate sulfur scarcity through desulfonation of various alkanesulfonates releasing sulfite which is incorporated into sulfur-containing biomolecules. This two-component monooxygenase system utilizes flavin as a substrate with the SsuE enzyme supplying reduced flavin to SsuD. It is unclear what structural properties of flavin reductases of two-component systems dictate catalysis. The SsuE enzyme undergoes a tetramer to dimer oligomeric switch in the presence of FMN. Oligomeric state changes are common in flavin reductases but the roles and regulation of the quaternary structural changes have not been evaluated. Intriguingly, the flavin reductases of two-component systems contain π-helices located at the tetramer interface. π-Helices are generated by a single amino acid insertion in an established α-helix to confer an evolutionary advantage.
Substitution of the π-helix insertional residue in SsuE (Tyr118) generated FMN-bound Y118A and Y118S SsuE variants, unlike the wild-type enzyme which is flavin-free. Structural and kinetic analyses were performed on these canonical flavoprotein variants and on the flavin-free variants (Y118F and ΔY118 SsuE) to understand the roles of π-helix in SsuE. We further investigated the structural effects of perturbing the π-helix in SsuE by solving the three-dimensional structures of Y118A SsuE in the oxidized form, when reduced, and in the apo form. Our findings show the π-helix is vital in the functioning of SsuE as a flavin reductases of two-component systems. In combination, the studies suggested the π-helix in SsuE promotes structural stability through hydrogen bonding and π-stacking interactions, and facilitates the transfer of reduced flavin to SsuD through protein-protein interactions. The π-helix may regulate the oligomeric changes in SsuE upon flavin binding and could be vital in controlling dioxygen reactivity. We also report herein that reduced flavin transfer from SsuE to SsuD occurs through a channeling mechanism facilitated by conformational changes in the flexible loop of SsuD. Overall, these studies unravel the evolutionary role of the π-helices in defining oligomeric state changes and in the transfer of reduced flavin in two-component monooxygenase systems.||en_US