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 |
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Authors: | Carolyn Shirey Somayesadat Badieyan Pablo Sobrado |
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Institution: | From the ‡Department of Biochemistry and ;the §Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061 |
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Abstract: | 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 N5-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. |
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Keywords: | Aspergillus Flavin Molecular Dynamics Mutagenesis Siderophores C4a-hydroperoxyflavin N-Hydroxylating SidA Flavin-dependent Monooxygenase NADP(H) Role |
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