Cysteine dioxygenase (CDO) structurally resembles cupin enzymes that use a 3-His/1-Glu coordination scheme. However, the glutamate ligand is substituted with a cysteine (Cys93) residue, which forms a thioether bond with tyrosine (Tyr157) under physiological conditions. The reversion variant, C93E CDO, was generated in order to reestablish the more common 3-His/1-Glu metal ligands of the cupin superfamily. This variant provides a framework for testing the structural and functional significance of Cys93 and the cross-link in CDO. Although dioxygen consumption was observed with C93E CDO, it was not coupled with l-cysteine oxidation. Substrate analogues (d-cysteine, cysteamine, and 3-mercaptopropionate) were not viable substrates for the C93E CDO variant, although they showed variable coordinations to the iron center. The structures of C93E and cross-linked and non-cross-linked wild-type CDO were solved by X-ray crystallography to 1.91, 2.49, and 2.30 Å, respectively. The C93E CDO variant had similar overall structural properties compared to cross-linked CDO; however, the iron was coordinated by a 3-His/1-Glu geometry, leaving only two coordination sites available for dioxygen and bidentate l-cysteine binding. The hydroxyl group of Tyr157 shifted in both non-cross-linked and C93E CDO, and this displacement prevented the residue from participating in substrate stabilization. Based on these results, the divergence of the metal center of cysteine dioxygenase from the 3-His/1-Glu geometry seen with many cupin enzymes was essential for effective substrate binding. The substitution of Glu with Cys in CDO allows for a third coordination site on the iron for bidentate cysteine and monodentate oxygen binding.
The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.
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.
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