Elucidating the flavin reductase mechanism in the alkanesulfonate monooxygenase system from Escherichia coli

Abstract

The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP+ as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the Km value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductases reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin. Rapid reaction kinetic analyses of SsuE were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. A weak charge-transfer complex between the flavin and pyridine nucleotide was identified in these studies. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following the binding of FMN and NADPH to SsuE (MC-1, Michaelis complex), an initial fast phase (241 s-1) corresponds to the interaction of NADPH with FMN (CT-1, charge-transfer complex). The second phase is a slow conversion (11 s-1) to form a charge-transfer complex of reduced FMNH2 with NADP+ (CT-2). The conversion of CT-1 to CT-2 is the step representing electron transfer from the pyridine nucleotide to the flavin. The third step (19 s-1) is the decay of the charge-transfer complex to the Michaelis complex of SsuE with bound products (MC-2). Results from isotope studies with the [4(R)-2H]NADPH substrate demonstrates the rate-limiting step in flavin reduction is electron transfer from NADPH to FMN. In addition, electron transfer is inhibited at high flavin concentrations, further implicating this step as rate-limiting. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins. Based on the steady-state and pre-steady-state kinetic analyses of SsuE, a reaction mechanism has been elucidated for the flavin reductase catalyzed reaction in the alkanesulfonate monooxygenase system.