Crystal Structure of Escherichia coli SsuE: Defining a General Catalytic Cycle for FMN Reductases of the Flavodoxin-like Superfamily

The Escherichia coli sulfur starvation utilization (ssu) operon includes a two-component monooxygenase system made up of an NADPH-dependent FMN reductase, SsuE, and a monooxygenase, SsuD. SsuE is part of the flavodoxin-like superfamily, and we report here the crystal structures of its apo, FMN-bound and FMNH₂-bound forms at ~2 Å resolution. In the crystals, SsuE forms a tetramer that is a dimer of dimers similar to those of seen for homologous FMN-reductases, quinone reductases, and the WrbA family of enzymes. A π-helix present at the tetramer building interface is unique to the reductases from two component monooxygenase systems. Analytical ultracentrifugation studies of SsuE confirm a dimer-tetramer equilibrium exists in solution with FMN binding favoring the dimer. As the active site includes residues from both subunits, at least a dimeric association is required for the function of SsuE. The structures show that one FMN binds tightly in a deeply held site which makes available a second binding site, in which either a second FMN or the nicotinamide of NADPH can bind. The FMNH₂-bound structure shows subtle changes consistent with its weaker binding compared to FMN. Combining this information with published kinetics studies, we propose a general catalytic cycle for two-component reductases of the flavodoxin-like superfamily, by which the enzyme can potentially provide FMNH₂ to its partner monooxygenase by different routes depending on the FMN concentration and the presence of a partner monooxygenase.This is an author's peer-reviewed final manuscript, as accepted by the publisher

Exposing the Alkanesulfonate Monooxygenase Protein–Protein Interaction Sites

The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein–protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen–deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein–protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein–protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation