The complex mechanistic properties of alkanesulfonate monooxygenase (SsuD) provide a particular challenge for identifying catalytically relevant amino acids. In response, a joint computational and experimental study was conducted to further elucidate the SsuD mechanism. Extensive unbiased molecular dynamics (MD) simulations were performed for six SsuD systems: (1) substrate-free, (2) bound with FMNH2, (3) bound with a C4a-peroxyflavin intermediate (FMNOO-), (4) bound with octanesulfonate (OCS), (5) co-bound with FMNH2 and OCS, and (6) co-bound with FMNOO- and OCS. A previous theoretical study suggested that salt bridges between Arg297 and Glu20 or Asp111 initiated conformational changes critical for catalysis. However, our MD simulations and steady-state kinetic experiments did not corroborate this result. Similar kcat/Km values for both the E20A and D111A SsuD variants to wild-type SsuD suggest that the salt bridges are not critical to the desulfonation mechanism. Instead, the predicted role of Arg297 is to favorably interact with the phosphate group of the reduced flavin. Concomitantly, Arg226 functioned as a "protection" group shielding FMNOO- from bulk solvent and was more pronounced when both FMNOO - and OCS were bound. The stabilization of FMNOO- through electrostatic interactions with Arg226 would properly position the C4a peroxy group for the proposed nucleophilic attack on the sulfur of octanesulfonate.
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