Investigating Structural Features Utilized by Two‐component Flavin‐dependent Systems

Abstract

Many enzymes exist in different oligomeric states which serves to regulate enzyme activity, integrate different pathways, expose new active sites at the interface of the interacting subunit, and enhance protein stability. Two‐component FMN‐dependent systems expressed in bacteria during times of limited sulfur availability regulate enzyme activity through the coordination of oligomeric changes of the reductase with reduced flavin transfer to the monooxygenase. The alkanesulfonate monooxygenase system is ubiquitous in bacteria, the reductase (SsuE) and the monooxygenase (SsuD) catalyze the desulfonation of alkanesulfonates to the corresponding aldehyde and sulfite. Pseudomonas aeruginosa has a more complex mechanism for acquiring sulfur. In addition to the SsuE/SsuD system, the organism also encodes a flavin‐dependent reductase (MsuE) and two monooxygenases (MsuC/MsuD) that together convert methanesulfinate to formaldehyde and sulfite. In the alkanesulfonate monooxygenase systems, the FMN reductases exist in different oligomeric states. SsuE exists as a tetramer (dimer of dimers) but can shift to a dimer in the presence of flavin and/or substrate. Coordinated structural dynamics of the FMN reductases and monooxygenase enables efficient synchronization of labile flavin transfer and catalysis. However, the unique structural features that integrates the oligomeric state of the reductase with the mode of flavin transfer to the monooxygenase has not been fully explored.Protein‐protein interactions between the FMN reductases and monooxygenases prevent the unproductive oxidation of reduced flavin. Studies were performed to investigate the interaction sites and oligomeric alterations critical for the transfer of reduced flavin from the reductase to the partner monooxygenase. SsuE and MsuE share 30% amino acid identity and SsuD and MsuD have 60% amino acid sequence identity, implying that although the enzymes utilize different substrates, they share similar structural strategies to regulate flavin transfer. Results from fluorescence spectroscopy show a high binding affinity between SsuE/SsuD and MsuE/MsuD. The high binding affinity observed ensures the safe transfer of reduced flavin from the reductase to the monooxygenase. Protein‐protein interactions sites were identified using hydrogen‐deuterium exchange with mass spectrometry (HDX‐MS) to evaluate flavin transfer in the alkanesulfonate monooxygenase systems (SsuE/SsuD and MsuE/MsuD). The interaction sites in SsuE are located at the dimer‐dimer interface and are close to the active site, while the monooxygenase interactions sites are conserved across SsuD enzymes from different bacterial sources. Comparable protein‐protein interaction sites observed in the alkanesulfonate monooxygenase system (SsuE/SsuD) were identified in the methanesulfinate monooxygenase system (MsuE/MsuD). Taken together, oligomeric changes of the flavin reductase regulates the transfer of flavin and the dimer‐dimer interface houses the sites for protein‐protein interactions with the monooxygenase facilitating catalysis.