Elucidating the Flavin Reductase Mechanism in a Two‐ component Monooxygenase System from Escherichia coli

Abstract

In Escherichia coli, sulfate or cysteine starvation induces the synthesis of a set of proteins which are responsible in acquiring sulfur from alternate sources. An FMN reductase (SsuE) and an FMNH2-utilizing monooxygenase (SsuD) are associated with sulfur acquisition from alkanesulfonates. SsuE catalyzes the reduction of FMN by NADPH to form FMNH2, and FMNH2 is then transferred to SsuD which converts the alkanesulfonate to aldehyde and sulfite in the presence of O2. The reductive reaction of SsuE has been extensively studied by kinetic analyses with single enzyme and coupled enzyme assays to define the reaction mechanism. The steady-state kinetic results indicate that the mechanism of SsuE is an ordered sequential mechanism. Interestingly, in the presence of SsuD and alkanesulfonate the kinetic mechanism is altered to a rapid equilibrium ordered mechanism. Rapid reaction kinetic analyses were performed to investigate the individual steps involved in flavin reduction. Two charge transfer complexes (CT) were generated during the reduction of FMN by NADPH, and characterized as FMN associated with NADPH (CT1), and FMNH2 with NADP+ (CT2). The rate of each charge transfer complex was studied by varying NADPH concentrations. The results indicate a rapid formation of CT1 that is NADPH-dependent, followed by a slower conversion of CT1 to CT2 that is not dependent on NADPH. Current studies are focused on the isotopic effect on flavin reduction by SsuE with (4R)-[2H]NADPH to determine the rate-limiting step in the overall reaction. Based on the kinetic data, a mechanistic scheme has been developed for flavin reduction by SsuE. This research is supported by Auburn University