RESUMO
A subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a π-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved α4 helix. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintained the π-π stacking interactions at the tetramer interface and had kinetic parameters similar to those of wild-type SsuE. Substitution of the π-helical residue (Tyr118) to Ala or Ser transformed the enzymes into flavin-bound SsuE variants that could no longer support flavin reductase and desulfonation activities. These variants existed as dimers and could form protein-protein interactions with SsuD even though flavin transfer was not sustained. The ΔY118 SsuE variant was flavin-free as purified and did not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of ΔY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE π-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD.
