Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 45, Pages 32440-32448Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.487181
Keywords
Aspergillus; Flavin; Molecular Dynamics; Mutagenesis; Siderophores; C4a-hydroperoxyflavin; N-Hydroxylating; SidA; Flavin-dependent Monooxygenase; NADP(H) Role
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Funding
- National Science Foundation [MCB 1021384]
- Virginia Tech Biodesign and Bioprocessing Center
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1021384] Funding Source: National Science Foundation
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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.
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