Catalytic Mechanism of a Flavin-Dependent Alkanesulfonate Monoxygenase from Escherichia coli

Abstract

The flavin-dependent alkanesulfonate monooxygenase (SsuD) catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2 provided by an FMN reductase (SsuE). The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics, steady-state kinetics, and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 compared to FMN. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate. The conserved putative active site His228 residue was proposed as a catalytic base in the desulfonation reaction by SsuD. In this study, this conserved residue was replaced by alanine, asparatate and lysine (H228A, H228D, and H228K). The catalytic efficiencies (kcat/Km) of His228A, H228D, and H228K SsuD with octanesulfonate as substrate were 50-200 fold lower than wild-type, suggesting the involvement of His228 in the desulfonation reaction by SsuD. Stopped-flow kinetic analyses indicated that the His228 residue is not directly involved in the reaction between the C4a-(hydro)peroxyflavin and alkanesulfonate substrate. The pH dependence on the kcat and kcat /Km values of wild-type and H228A SsuD were similar, suggesting His228 is not a catalytic base in catalytic steps involving the octanesulfonate substrate. The activity for H228A SsuD could be partially rescued with exogenous imidazole at increasing pH, indicating the deprotonated form of histidine exerts a functional role. Further analyses indicates that this residue is more likely indirectly participating in the catalysis by stabilizing and properly orientating another amino acid, that is directly involved in the desulfonation reaction.