Abstract 2153 Exploring the Quaternary States of the Class C TC-FMO SfnG

Sulfur is a crucial element for life, constituting less than 1% of an organism's biomass yet playing essential roles within metabolism. Many microorganisms typically uptake sulfate and sulfite molecules from their environment. However, when depleted, alternative methods for sulfur uptake and conversion are employed. Within Pseudomonas fluorescens, a set of genes is expressed that allows for the utilization of alkanesulfonates to generate sulfite in this limiting condition. The proteins expressed are two-component flavin-dependent monooxygenases (TC-FMOs), involved in the desulfonation of the organosulfur compound, and a flavin mononucleotide (FMN) reductase essential for the generation of reduced FMN. The first step in this pathway is SfnG, a TC-FMO that acts upon dimethylsulfone to produce methanesulfinate. This compound is further acted on by enzymes in this pathway to generate sulfite. DLS and SEC-MALS studies of P. fluorescens SfnG revealed a quaternary state that shifted from a tetramer to a dimer in the presence of high salt, with restoration of the tetramer with the addition of oxidized FMN and DMSO2. A 2.6-Å unliganded structure and a 1.75-Å ternary complex of SfnG revealed a unique quaternary structure compared to other class C TC-FMOs. The ternary complex also showed the typically disordered lid region of SfnG to be structured. Comparing the unliganded structure and ternary complex highlights how the ordered lid region contributes to stabilizing the tetramer complex. Structural analysis suggests SfnG may be a class C TC-FMO utilizing N5-(hydro)peroxyflavin in its chemistry. The active site reveals a loop connecting the putative dioxygen binding site to a latch area for possible substrate release. Overall, these results contribute to our understanding of the varied quaternary states of TC-FMOs involved in C–S bond cleavage and provide another example for studying N5-(hydro)peroxyflavin chemistry. Funding provided by the National Science Foundation Award 1807480. Acknowledgement to the Biophysical Instrumentation Core at UMass Boston.