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.