Characterization of the flavin dependent dimethylsulfone monooxygenase SfnG from Pseudomonas fluorescens

Abstract

Sulfur is one of the essential elements of life, being involved in pathways to generate sulfide, which is used downstream for amino acid, metabolite, and signaling molecule synthesis. Inorganic sulfate and sulfite are the typical sources of sulfur for these bacteria; however, limiting sulfate/sulfite conditions require bacteria to use alternative sulfur sources. Within Pseudomonas fluorescens a set of genes are expressed that allow them to utilize 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, which is further acted on by enzymes in this pathway to generate sulfite. Current work has structurally characterized SfnG through X‐ray crystallography with the ligands FMN and DMSO2 to 1.75‐Å resolution and without ligands to 2.6‐Å resolution. The characterization of SfnG elucidated residues predicted to be important for binding of DMSO2 and FMN within the active site. Further structural analysis showed the active site of SfnG is enclosed by a lid region which is disordered without substrates bound, and the structural arrangement of ligands and the predicted oxygen binding site is in support of SfnG utilizing the recently proposed N5‐peroxyflavin intermediate. Further analysis of SfnG has revealed an interesting quaternary state reliant on substrate binding. The quaternary structure organization is different from other TC‐FMO’s involved in C–S bond cleavage, which was further explored through FMN binding studies. These results will contribute towards our knowledge of TC‐FMOs involved in C–S bond cleavage.