Abstract 2136 Crystallization and biophysical analysis of short to medium-chain alkanesulfonate monooxygenase models reveals ligand dependent stabilization and dimer-tetramer dynamics

The 5th most abundant element in life is sulfur, and the element takes many forms that provide vital roles throughout the cell. From organosulfur compounds such as cysteine, glutathione, and sulfoquinovose, sulfur is widely utilized. The sulfur cycle is the flow of chemical forms of sulfur as they change in composition across and between systems both living and environmental, and one important junction of the cycle is the breakdown of organosulfur compounds for living beings into utile ones through various pathways. In Pseudomonads, the assimilation of these forms of sulfur happens when the bacteria are deprived of typical usable sources of sulfur-containing biomolecules, leading to the sulfur starvation response. The bacteria respond by uptaking and transforming alternative organosulfur compounds into sources of sulfur, carbon and energy via two-component flavin-dependent monooxygenases (TC-FMOs) that are capable of cleaving stable C–S bonds to produce sulfite. The TC-FMO MsuD is the final enzyme in a pathway that breaks down methanesulfonate, a molecule that has had its natural levels in the environment greatly increased by anthropogenic sources. Here we present the crystal structure of MsuD from Pseudomonas fluorescens in complex with FMN, and we contrast this information to the previously reported structures of MsuD containing both FMN and methanesulfonate. The oligomeric state and thermal stability of MsuD are explored using both light scattering and intrinsic protein fluorescence. The data presented show that both stability and the quaternary state equilibrium are dependent on ligand binding. These results reveal how the dynamic dimer-tetramer equilibrium of MsuD shifts as part of the greater reaction scheme and is likely important to the catalytic cycle. The role of FMN beyond catalysis and into quaternary structure stabilization suggests how the redox balance of the cell could alter the MsuD oligomeric state. In addition, this dimer-tetramer change in morphology and the ligand binding that drives it is not observed in other TC-FMOs. By determining the factors that drive this change, a more accurate picture of MsuD and similar functions in other TC-FMOs can become a basis for further research. Funding support from the National Science Foundation under grant number 1807480.