BIOL 178

The three-dimensional structure of a similar monooxygenase, bacterial luciferase, has been extensively studied and the catalytically relevant residues identified (3). The role of Cys106, located in the active site of bacterial luciferase, has been extensively studied (4). Mutation of this residue to valine was shown to destabilize the C4a-hydroperoxyflavin intermediate formed in the reaction (5). Although SsuD shares very little amino acid sequence identity with bacterial luciferase, their overall three-dimensional structures are similar. SsuD contains a cysteine residue in a similar spatial arrangement to that of Cys106 in luciferase. This putative catalytic thiol of SsuD, Cys54, was mutated to alanine and serine (C54A and C54S SsuD, respectively) to determine the role of this residue in catalysis. Steady-state kinetic parameters for the C54A SsuD variant resulted in a complete loss of activity, while the C54S mutation caused a two-fold decrease in activity. A dissociation constant, K_d, for FMN or FMNH₂ was obtained by monitoring the decrease in fluorescence emission intensity at 340 nm. Both mutations caused a five-fold decrease in the K_d for FMNH₂, but little change for FMN binding. Rapid reaction kinetic analyses were also performed to characterize the catalytic step affected by the Cys substitution. The reported results further define the role of the putative reactive thiol located in the active site of the alkanesulfonate monooxygenase enzyme.