Siderophore A (SidA) is an ornithine hydroxylase that belongs to the class B flavin monooxygenase family of enzymes. SidA catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Previous structural analysis revealed that R144, a residue adjacent to the FAD, undergoes redox-dependent conformational changes. In order to determine the role of R144 in catalysis, site-directed mutagenesis was used to create the R144A enzyme. Steady-state kinetic measurements showed that the mutant variant R144A has a 20-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step responsible for the reduced steady-state activity. The data are consistent with flavin dehydration being the main step affected by the mutation. We proposed that R144 plays a role, at least in part, in providing the proper hydrogen bonding network for the elimination of the hydroxide from the flavin-hydroxide intermediate. Implication for a conserved mechanism in other flavin-dependent enzymes is discussed.
Role of R144 in the dehydration of the C4a-hydroxyflavin in SidA