Structural Properties in the FMN Reductase of the Alkanesulfonate Monooxygenase System that Dictate Oligomeric States and Catalysis

The alkanesulfonate monooxygenase system is expressed in many bacteria when sulfur is limiting to enable the organism to utilize a diverse range of alkanesulfonates. This system is comprised of a NADPH‐dependent FMN reductase (SsuE) that reduces flavin and transfers it to the monooxygenase enzyme (SsuD) for the desulfonation of alkanesulfonates 1 . The SsuE enzyme exists in different oligomeric states, but it is unclear how these oligomeric states relate to catalysis. A conserved π‐helix is located in the tetramer interface of SsuE which results from the insertion of Tyr118 in the α4‐helix. This Tyr118 residue forms a hydrogen bond with the carboxyl group of Ala78 across the tetramer interface to enhance the protein architecture 2 . Variants of Y118 SsuE were generated to evaluate the role of the π‐helix in SsuE. Interestingly, the Y118A and Y118S SsuE variants were FMN‐bound as purified, while the Y118F and ΔY118 SsuE variants were flavin‐free. The Y118 SsuE variants were able to effectively bind FMN giving similar K d values as wild‐type SsuE. This flavin affinity correlated with the ability of Y118 SsuE to reduce flavin with k cat / K m values similar to that of wild‐type SsuE in flavin reductase assays. While desulfonation activity was observed with the Y118F SsuE variant, there was no activity in the coupled assays monitoring SsuD desulfonation with the Y118A, Y118S and the ΔY118 variants of SsuE. This would indicate that the Tyr118 is vital for the transfer of FMNH 2 to SsuD. The results from these studies suggest that multiple non‐covalent interactions are important for the structural stability of SsuE. These findings provide insights on the role of Tyr118 in SsuE. 1. Eichhorn, E. et al. (1999) J. Biol. Chem. 274 , 26639‐46. 2. Driggers, C. M. et al (2014) Biochem. 53 , 3509‐19.