Functional Roles of Conserved Active Site Amino Acids in the Desulfonation Reaction Catalyzed by the Alkanesulfonate Monooxygenase from Escherichia coli

Abstract

The flavin-dependent alkanesulfonate monooxygenase (SsuD) catalyzes the oxidation of 1-substituted alkanesulfonates producing sulfite and a corresponding aldehyde in the presence of O2 and FMNH2. The reduced flavin is supplied by the NAD(P)H-dependent FMN reductase, SsuE. The goal of these studies was to investigate the roles of conserved amino acids located in or around the postulated active site of SsuD through various kinetic and substrate binding experiments. A conserved cysteine residue located at position 54 in SsuD is proposed to stabilize the C4a-(hydro)peroxyflavin intermediate formed during the desulfonation reaction. This conserved residue was replaced with either serine or alanine (C54S and C54A). The catalytic efficiencies (kcat/Km) obtained for C54S and C54A with octanesulfonate were 3-fold greater and 6-fold lower than wild-type SsuD, respectively. Results from fluorescence titration experiments with octanesulfonate suggest that the Cys54 residue is not involved in FMNH2 binding; however, the binding of octanesulfonate substrate was affected by substitution with Ser or Ala. Stopped-flow kinetic analysis gave evidence that the polar nature of the amino acid residue at position 54 was key to a high accumulation of the C4a-(hydro)peroxyflavin intermediate. These results suggest that the cysteine residue located at position 54 in SsuD is involved in the stabilization of the C4a-(hydro)peroxyflavin intermediate formed during the desulfonation reaction catalyzed by SsuD. When SsuD was first purified an aberrant mutation of Arg297 to Cys abolished all activity. It was postulated that this arginine residue was located on a flexible loop and mutation to cysteine would cause a disulfide bond to form with Cys54. This flexible loop containing the conserved Arg297 residue is proposed to close over the active site to protect intermediates during catalysis. This conserved residue was replaced with either lysine or alanine (R297K and R297A). The R297K SsuD variant exhibited a 30-fold decrease in the kcat/Km value compared to wild-type. In contrast, the R297A SsuD variant had no detectible activity, even at higher enzyme concentrations. Fluorescence titrations with FMNH2 showed a minor 3-fold increase in the dissociation constant (Kd) of FMNH2 for both SsuD variants; however, octanesulfonate was unable bind. Stopped-flow kinetic experiments show the formation of the C4a-(hydro)peroxyflavin intermediate with both R297K and R297A SsuD variants. Rapid-reaction kinetic traces of the R297 SsuD variants in the presence of SsuE showed an altered step in flavin oxidation compared to wild-type. Proteolytic digestions suggest that the Arg297 located on the postulated mobile loop is important for protecting the SsuD enzyme from proteolysis. These studies give evidence for the stabilization of the C4a-(hydro)peroxyflavin intermediate by Cys54 and the protection of substrates and/or intermediates during the desulfonation reaction by Arg297.