Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation.

Bacteria have evolved to utilize alternative organosulfur sources when sulfur is limiting. The SsuE/SsuD and MsuE/MsuD enzymes expressed when sulfur sources are restricted, are responsible for providing specific bacteria with sulfur in the form of alkanesulfonates. In this study, we evaluated why two structurally and functionally similar FMNH2-dependent monooxygenase enzymes (MsuD and SsuD) are needed for the acquisition of alkanesulfonates in some bacteria. In desulfonation assays, MsuD was able to utilize the entire range of alkanesulfonates (C1-C10). However, SsuD was not able to utilize smaller alkanesulfonate substrates. Interestingly, SsuD had a similar binding affinity for methanesulfonate (MES) (15 ± 1 µM) as MsuD (12 ± 1 µM) even though SsuD was not able to catalyze the desulfonation of the MES substrate. SsuD and MsuD showed decreased proteolytic susceptibility in the presence of FMNH2 with MES and octanesulfonate (OCS). Tighter loop closure was observed for the MsuD/FMNH2 complex with MES and OCS compared to SsuD under comparable conditions. Analysis of the SsuD/FMNH2/MES structure using accelerated molecular dynamics simulations found three different conformations for MES, demonstrating the instability of the bound structure. Even when MES was bound in a similar fashion to OCS within the active site, the smaller alkane chain resulted in a shift of FMNH2 so that it was no longer in a position to catalyze the desulfonation of MES. The active site of SsuD requires a longer alkane chain to maintain the appropriate architecture for desulfonation.

Substrate-Dependent Mobile Loop Conformational Changes in Alkanesulfonate Monooxygenase from Accelerated Molecular Dynamics.

Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH2), C4a-peroxyflavin intermediate (FMNOO-), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: "open", "closed", and "semiclosed". The substrate-free SsuD system possessed a wide opening capable of providing full access for substrates to enter the active site. Upon binding FMNH2, SsuD adopts a closed conformation that would prevent unproductive oxidation reactions in the absence of OCS. Two salt bridges, Asp111-Arg263 and Glu205-Arg271, were identified as particularly important in maintaining the closed conformation. Experimental substitution of Arg271 to Ala did not alter the catalytic activity, but the variant in the presence of reduced flavin was more susceptible to proteolytic digestion compared to wild-type. With both FMNH2 and OCS bound in SsuD, a second conformation was formed dependent upon a favorable π-π interaction between His124 and Phe261. Accordingly, there was no observed activity with the F261W SsuD variant in steady-state kinetic assays. This semiclosed conformation may be more appropriate for accepting O2 into the binding pocket and/or may properly orient the active site for the ensuing oxygenolytic cleavage. Finally, simulations of SsuD simultaneously bound with FMNOO- and OCS found an open mobile loop region that suggests alternative flavin intermediates may participate in the reaction mechanism.