Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

SidA (siderophore A) is a flavin-dependent N -hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus . SidA catalyzes the NADPH- and oxygen-dependent formation of N 5 -hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP + is the last product to be released. The presence of NADP + is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP + to slide into position for stabilization of the C4a-hydroperoxyflavin. Background: SidA is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine and is essential for virulence in Aspergillus fumigatus . Results: Mutation of Ser-257 leads to destabilization of the C4a-hydroperoxyflavin and a faster hydride transfer step. Conclusion: Ser-257 is important for positioning NADP + in the correct orientation for stabilization of the C4a-hydroperoxyflavin. Significance: The results support a sliding mechanism for NADP(H) in SidA.